Therapeutic and diagnostic methods relating to cancer stem cells

ABSTRACT

The present invention relates in part to the discovery of genes that are deregulated in cancer stem cells (e.g., melanoma stem cells). In some aspects, methods for treating individuals having melanoma are provided; the methods involve modulating (e.g., inducing, inhibiting, etc.) the activity of the cancer stem cell associated genes. In other aspects, cell surface genes that are upregulated in melanoma stem cells are targeted for the selective isolation, detection, and killing of cancer stem cells in melanoma. Other aspects of the invention relate to reagents, arrays, compositions, and kits that are useful for diagnosing and treating melanoma.

RELATED APPLICATION

This application claims priority under 35 U.S.C. § 119 from U.S.provisional application Ser. No. 61/114,490, filed Nov. 14, 2008, thecontents of which are incorporated herein in their entirety.

FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under Grant5R01CA113796-03 from the National Cancer Institute. The Government hascertain rights to this invention.

FIELD OF INVENTION

The present invention relates in part to methods for treatingindividuals having cancer. The methods involve modulating, e.g.,inducing or inhibiting, the activity of genes that are deregulated incancer stem cells. In some aspects, cell surface genes that areupregulated in cancer stem cells are targeted for selective isolation,detection, or killing of cancer stem cells in melanoma. Other aspects ofthe invention relate to reagents, arrays, compositions, and kits thatare useful for diagnosing and treating cancer.

BACKGROUND OF INVENTION

Self-renewing cancer stem cells (CSCs) initiate tumours and drivemalignant progression by generating and supporting replication of moredifferentiated non-stem cell progeny. (M. Al-Hajj, et al., Proc NatlAcad Sci USA 100 (7), 3983 (2003); D. Bonnet and J. E. Dick, Nat Med 3(7), 730 (1997); C. A. O'Brien, et al., Nature 445 (7123), 106 (2007);L. Ricci-Vitiani, et al., Nature 445 (7123), 111 (2007); S. K. Singh, etal., Nature 432 (7015), 396 (2004); T. Schatton and M. H. Frank, Pigmentcell & melanoma research 21 (1), 39 (2008)). The mechanisms by whichCSCs cause tumour formation and growth and the potential role ofCSC-specific differentiation plasticity in tumourigenicity are currentlyunknown. We recently identified a subpopulation of CSC based onexpression of the chemoresistance mediator ABCB5 (ATP-binding cassette,sub-family B (MDR/TAP), member 5) (N. Y. Frank, A et al., Cancer Res 65(10), 4320 (2005); Y. Huang, et al., Cancer Res 64 (12), 4294 (2004))within human malignant melanoma (T. Schatton, et al., Nature 451 (7176),345 (2008)), a highly aggressive and drug-resistant cancer. (T. Schattonand M. H. Frank, Pigment cell & melanoma research 21 (1), 39 (2008); L.Chin, L. A. Garraway, and D. E. Fisher, Genes Dev 20 (16), 2149 (2006).)ABCB5⁺ Malignant Melanoma Initiating Cells (MMIC) correlate withclinical disease progression and can be specifically targeted toabrogate tumour growth. (T. Schatton, et al., Nature 451 (7176), 345(2008)). Consistent with these findings, the ABCB5 gene is alsopreferentially expressed by in vitro self-renewing melanoma minoritypopulations (G. I. Keshet, et al., Biochem Biophys Res Commun 368 (4),930 (2008)) and by melanoma cell lines of metastatic as opposed toprimary, radial growth phase tumour origin (J. F. Sousa and E. M.Espreafico, BMC cancer 8, 19 (2008)).

SUMMARY OF INVENTION

The present invention relates in part to the discovery that a number ofgenes (referred to herein as CSC-associated genes) are deregulated incancer stem cells. In some aspects, the invention relates to diagnosticarrays and methods for detecting cancer in an individual based on theexpression of CSC-associated genes. In other aspects, the inventionrelates to methods useful for treating individuals having melanoma basedon modulating the expression and/or activity of CSC-associated genes.Compositions and kits that are useful for the foregoing methods are alsodisclosed.

The invention, in some aspects, provides methods for diagnosing cancerin an individual. In some aspects, the methods involve determining anexpression level of a cancer stem cell (CSC)-associated gene in Table 5in a test sample from the individual and comparing the expression levelof the CSC-associated gene to a reference value, wherein results of thecomparison are diagnostic of cancer. In some embodiments, the cancer ismelanoma, breast cancer, prostate cancer, colon cancer or renal cancer.In some embodiments, the test sample is a tissue biopsy. In someembodiments, the test sample is a skin biopsy. In some embodiments, thetest sample is a sample of the cancer, such as a tumor biopsy. In someembodiments, the methods involve updating a patient record for theindividual to indicate the diagnostic result of the comparison. In someembodiments, determining comprises detecting in the test sample a mRNAthat is encoded by the CSC-associated gene. In some embodiments,determining comprises detecting in the test sample a polypeptide that isencoded by the CSC-associated gene. In certain embodiments, detectingcomprises nucleic acid hybridization or nucleic acid amplification. Inspecific embodiments, the nucleic acid amplification is real-time RT-PCRor RT-PCR. In one embodiment, the nucleic acid hybridization isperformed using a nucleic acid array. In certain other embodiments,detecting comprises immunodetection of the polypeptide. In oneembodiment, the immunodetection comprises an Enzyme-Linked ImmunosorbentAssay (ELISA). In one embodiment, the immunodetection comprises anantibody array. In one embodiment, the immunodetection comprisesimmunohistochemistry.

In some embodiments of the methods, the reference value is theexpression level of the CSC-associated gene in a non-cancer referencesample, and if the expression level of the CSC-associated gene in thetest sample is about equal to the expression level of the CSC-associatedgene in the non-cancer reference sample, then the comparison does notindicate cancer.

In some embodiments of the methods, the reference value is theexpression level of the CSC-associated gene in a cancer referencesample, and if the expression level of the CSC-associated gene is aboutequal to the expression level of the CSC-associated gene in the cancerreference sample, then the comparison indicates cancer.

In some embodiments of the methods, the CSC-associated gene is in Table1 or 8 and the reference value is the expression level of theCSC-associated gene in a non-cancer reference sample, and if theexpression level of the CSC-associated gene in the test sample issignificantly higher than the expression level of the CSC-associatedgene in the non-cancer reference sample, the comparison indicatescancer.

In some embodiments of the methods, the CSC-associated gene is in Table1 or 8 and the reference value is the expression level of theCSC-associated gene in a cancer reference sample, and if the expressionlevel of the CSC-associated gene in the test sample is significantlylower than the expression level of the CSC-associated gene in the cancerreference sample, the comparison does not indicate cancer.

In some embodiments of the methods, the CSC-associated gene is in Table1 or 8 and the reference value is the expression level of theCSC-associated gene in a non-cancer reference sample, and if theexpression level of the CSC-associated gene in the test sample is atleast 10% higher than the expression level of the CSC-associated gene inthe non-cancer reference sample, the comparison indicates cancer.

In some embodiments of the methods, the CSC-associated gene is in Table2 or 7 and the reference value is the expression level of theCSC-associated gene in a non-cancer reference sample, and if theexpression level of the CSC-associated gene in the test sample issignificantly lower than the expression level of the CSC-associated genein the non-cancer reference sample, the comparison indicates cancer.

In some embodiments of the methods, the CSC-associated gene is in Table2 or 7 and the reference value is the expression level of theCSC-associated gene in a cancer reference sample, and if the expressionlevel of the CSC-associated gene in the test sample is significantlyhigher than the expression level of the CSC-associated gene in thecancer reference sample, the comparison does not indicate cancer.

In some embodiments of the methods, the CSC-associated gene is in Table2 or 7 and the reference value is the expression level of theCSC-associated gene in a non-cancer reference sample, and if theexpression level of the CSC-associated gene in the test sample is atleast 10% lower than the expression level of the CSC-associated gene inthe non-cancer reference sample, the comparison indicates cancer.

The invention, in some aspects, provides methods for isolating a cancerstem cell. In some aspects, the methods involve contacting a sample withan agent that binds a polypeptide, which is encoded by a CSC-associatedgene in Table 4 and expressed on the surface of the cancer stem cell,and isolating the agent from the sample. If the sample contains thecancer stem cell, the agent binds to the polypeptide on the surface ofthe cancer stem cell such that isolation of the agent from the sampleresults in isolation of the cancer stem cell. In some embodiments, theCSC-associated gene is selected from the group consisting of: ANK2,NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1,STX3, and TBC1D8. In some embodiments, the agent is an isolated peptidethat specifically binds the polypeptide on the surface of the cancerstem cell. In certain embodiments, the isolated peptide is an antibodyor antigen-binding fragment. In specific embodiments, the antibody orantigen-binding fragment is a monoclonal antibody, polyclonal antibody,human antibody, chimeric antibody, humanized antibody, single-chainantibody, F(ab′)₂, Fab, Fd, Fv, or single-chain Fv fragment. In someembodiments, the isolated peptide is bound to a solid support. In someembodiments, the isolated peptide is conjugated to a detectable label.In some embodiments, the detectable label is a fluorophore which may beselected from: FITC, TRITC, Cy3, Cy5, Alexa Fluorescent Dyes, andQuantum Dots. In some embodiments, the isolating comprises performingfluorescent activated cell sorting to isolate a cancer stem cell boundto a detectable label. In some embodiments, the cancer stem cell is froma melanoma, breast cancer, prostate cancer, colon cancer or renalcancer.

The invention, in some aspects, provides methods for treating anindividual having, or at risk of having, cancer. In some aspects, themethods involve administering a therapeutically effective amount of acomposition that induces the expression of a CSC-associated geneselected from the group set forth in Table 2 or 7. In some embodiments,the cancer is melanoma, breast cancer, prostate cancer, colon cancer orrenal cancer.

In some embodiments, the composition that induces the expression of aCSC-associated gene comprises an isolated plasmid that expresses theCSC-associated gene. In some embodiments, the isolated plasmid is in avirus capable of infecting the individual. In certain embodiments, thevirus is selected from adenovirus, retrovirus, lentivirus, andadeno-associated virus. In some embodiments, the isolated plasmidcomprises a cancer specific promoter operably linked to theCSC-associated gene.

The invention, in other aspects, provides methods for treating anindividual having, or at risk of having, cancer that involveadministering a therapeutically effective amount of a composition thattargets a product of a CSC-associated gene selected from the group setforth in Table 1 or 8. In some embodiments, the cancer is melanoma,breast cancer, prostate cancer, colon cancer or renal cancer. In someembodiments, the CSC-associated gene is selected from the group setforth in Table 4. In certain embodiments, the CSC-associated gene isselected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS,SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.

In some embodiments, the composition that targets a product of aCSC-associated gene comprises a small interfering nucleic acid thatinhibits expression of the CSC-associated gene. In some embodiments, thecomposition comprises an isolated plasmid that expresses the smallinterfering nucleic acid. In certain embodiments, the plasmid is in avirus. In specific embodiments, the virus is selected from adenovirus,retrovirus, lentivirus, and adeno-associated virus. In certainembodiments, the isolated plasmid comprises a cancer-specific promoteroperably linked to a gene encoding the small interfering nucleic acid.

In some embodiments, the composition that targets a product (e.g.,protein or RNA) of a CSC-associated gene comprises an isolated moleculethat selectively binds to a polypeptide encoded by the CSC-associatedgene. In certain embodiments, the isolated molecule is conjugated to atherapeutic agent. In specific embodiments, the isolated molecule is anantibody or antigen-binding fragment. In particular embodiments, theantibody or antigen-binding fragment is a monoclonal antibody,polyclonal antibody, human antibody, chimeric antibody, humanizedantibody, a single-chain antibody, F(ab′)₂, Fab, Fd, Fv, or single-chainFv fragment. In specific embodiments, the therapeutic agent is selectedfrom: a toxin, a small-interfering nucleic acid, and a chemotherapeuticagent. In one embodiment, the toxin is a radioisotope. In particularembodiments, the radioisotope is selected from the group consisting of:²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I,¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra and ²²³Ra. In someembodiments, the therapeutic agent is a small interfering nucleic acidthat inhibits expression of a CSC-associated gene. In some embodiments,the isolated molecule binds to the polypeptide and enters anintracellular compartment of a cancer stem cell of the cancer.

In some embodiments, the treatment methods involve determining theexpression level of the CSC-associated gene in the individual. Incertain embodiments, the methods involve comparing the expression levelof the CSC-associated gene to a reference value, wherein results of thecomparison are diagnostic of cancer in the individual. In specificembodiments, if the comparison results in a diagnosis of cancer in theindividual, the administering is performed. In one embodiment, thedetermining and the comparing are repeated at one or more intervalsafter the administering. In some embodiments, the administering isorally, intravenously, intrapleurally, intranasally, intramuscularly,subcutaneously, intraperitoneally, or as an aerosol.

The invention, in some aspects, provides methods of delivering atherapeutic agent to a cancer stem cell that involve contacting a cancerstem cell with an isolated molecule, which selectively binds to apolypeptide encoded by a CSC-associated gene selected from the group setforth in Table 4 and which is conjugated to a therapeutic agent, in aneffective amount to deliver the therapeutic agent to the cancer stemcell. In some embodiments, the CSC-associated gene is selected from thegroup consisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA,SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments,the isolated molecule is an antibody or antigen-binding fragment thatselectively binds the polypeptide. In some embodiments, the therapeuticagent is selected from: a toxin, a small-interfering nucleic acid, and achemotherapeutic agent. In one embodiment, the toxin is a radioisotope.In particular embodiments, the radioisotope is selected from the groupconsisting of: ²²⁵Ac, ²¹¹At, ²¹²Bi, ²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y,¹³¹I, ⁶⁷Cu, ¹²⁵I, ¹²³I, ⁷⁷Br, ¹⁵³Sm, ¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra and²²³Ra. In some embodiments, the therapeutic agent is a small interferingnucleic acid that inhibits expression of a CSC-associated gene. In someembodiments, the cancer stem cell is in vitro. In other embodiments, thecancer stem cell is in vivo.

In some aspects, the invention provides nucleic acid arrays consistingessentially of at least 2, at least 5, at least 10, at least 20, atleast 50, at least 100, at least 200, at least 300, or moreCSC-associated genes set forth in Table 5.

In some aspects, the invention provides polypeptide arrays consistingessentially of at least 2, at least 5, at least 10, at least 20, atleast 50, at least 100, or more polypeptides or immunogenic fragmentsthereof encoded by CSC-associated genes set forth in Table 1 or 8. Insome aspects, the invention provides antibody arrays consistingessentially of at least 2 or more different antibodies orantigen-binding fragments that selectively bind polypeptides encoded byCSC-associated genes set forth in Table 1 or 8.

In some aspects, the invention provides methods for stratifying apopulation comprising individuals having cancer. The methods involvedetermining expression levels of at least 2, at least 5, at least 10, atleast 20, at least 50, at least 100, at least 200, at least 300, or moreCSC-associated genes set forth in Table 5 and stratifying the populationbased on the expression levels.

In some aspects, the invention provides an isolated molecule thatselectively binds to a polypeptide encoded by a CSC-associated gene setforth in Table 4, and that is conjugated to a therapeutic agent. In someembodiments, the CSC-associated gene is selected from the groupconsisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB,SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments, thetherapeutic agent is selected from: a toxin, a small-interfering nucleicacid, and a chemotherapeutic agent.

In certain embodiments, the isolated molecule is an antibody orantigen-binding fragment. In certain embodiments, the antibody orantigen-binding fragment is a monoclonal antibody, polyclonal antibody,human antibody, chimeric antibody, humanized antibody, single-chainantibody, a F(ab′)₂, Fab, Fd, or Fv fragment. In certain embodiments,the isolated molecule is an isolated receptor ligand of the polypeptide.

The invention, in some aspects, provides compositions comprising any ofthe foregoing isolated molecules. In some embodiments, the compositionsinclude a pharmaceutically acceptable carrier.

The invention, in some aspects, provides pharmaceutical kits thatinclude a container housing any of the foregoing compositions andinstructions for administering the composition to an individual havingcancer.

Use of a composition of the invention for treating cancer is alsoprovided as an aspect of the invention.

A method for manufacturing a medicament of a composition of theinvention for treating cancer is also provided.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1I depict an analysis of vasculogenic/angiogenic pathways inhuman melanoma. FIG. 1a . is a graphical representation of pathwayactivation across ABCB5⁺ MMIC. Genes represented by nodes (dark graycircles, TRIO, MET, FLT1, PSEN1, NRP2, RHOA, PTK2, PIP5K3, KIAA1267,MLL, GABPA, ETS1, and CHD8) are overexpressed in ABCB5⁺ relative toABCB5⁻ human melanoma cells and those represented by black nodes areexpressed at lower levels, respectively. Black lines between genes showknown interactions. Known gene functions in vasculogenesis andangiogenesis, and genes known as relevant drug targets are annotated(dark gray lines). Gene relationships and figure layout are based onIngenuity Pathway Analysis and references are provided elsewhere in thetext. FIG. 1b . shows detection of vasculogenic/angiogenic pathwaymembers by RT-PCR in ABCB5⁺ MMIC. FIG. 1c . shows FLT1 (VEGFR-1) proteinexpression on ABCB5⁺ MMIC (top) and ABCB5⁻ melanoma cells (bottom) asdetermined by dual color flow cytometry using ABCB5 phenotype-specificcell gating, with mean percentages (mean±s.e.m., n=6 replicateexperiments) shown on the right. FIG. 1d . depicts representativeimmunofluorescence staining for CD144 expression (Texas red staining) byABCB5⁺ MMIC or ABCB5⁻ melanoma cell subpopulations prior to (t=0 h) andupon 48 h of culture (t=48 h) in the presence of 100 ng/ml VEGF¹¹, withnuclei counterstained with DAPI. Mean percentages (mean±s.e.m., n=3replicate experiments) of cells staining positively for CD144 in eachsample are shown on the right. FIG. 1e . shows representativeimmunofluorescence staining for CD144 expression (Texas red staining) bymelanoma cells cultured for 48 h (t=48 h) in the presence of 100 ng/mlVEGF as in above, but in the presence or absence of anti-FLT1 (VEGFR-1)blocking mAb or isotype control mAb. Nuclei are counterstained withDAPI. Mean percentages (mean±s.e.m., n=3 replicate experiments) of cellsstaining positively for CD144 in each sample are shown in the far rightpanel. FIG. 1f . shows tube formation detected by phase contrast lightmicroscopy of melanoma cells cultured for 24 h (t=24 h) in the presenceof 100 ng/ml VEGF and the presence or absence of anti-FLT1 (VEGFR-1)blocking mAb or isotype control mAb. Number of tubes/microscopy field(mean±s.e.m., n=3 replicate experiments) and tube length (μm)(mean±s.e.m., n=3 replicate experiments) are illustrated for thedifferent experimental conditions on the far right panels, respectively.FIG. 1g . shows the adipogenic differentiation potential of ABCB5⁺ andABCB5⁻ human melanoma cells (Oil Red O staining, nuclei arecounterstained with hematoxylin). FIG. 1h . shows the osteogenicdifferentiation potential of ABCB5⁺ and ABCB5⁻ human melanoma cells(Alizarin Red staining). FIG. 1i shows the myogenic differentiationpotential of ABCB5⁺ and ABCB5⁻ human melanoma cells. Absence of myogeninstaining (FITC, green) is detected in ABCB5⁺ or ABCB5⁻ human melanomacells (nuclei are counterstained with DAPI).

FIGS. 2A-2K depict an analysis of MMIC-driven in vivo vasculogenesis.FIG. 2a . shows conventionally-stained (H&E) sections of human melanomagrowing at melanoma cell injection site within human dermis of skinxenograft to NOD/SCID mouse. FIG. 2b . shows immunohistochemistry forhuman CD31 indicating angiogenic response at perimeter of melanomawithin human xenograft; broken line represents interface of tumournodule with dermal connective tissue. FIG. 2c . shows PAS (withdiastase) immunochemical staining of CD31-negative interior regions ofmelanoma xenograft revealing numerous anastomosing channels (inset islaminin immunohistochemistry indicating identical pattern). FIG. 2d .shows transmission electron micrographs of interior regions of melanomaxenograft; lumenal spaces containing blood products (erythrocytes) arelined by melanoma cells and associated basement membrane-likeextracellular matrix. FIG. 2e . shows the interior zone of melanomaxenograft derived from cells expressing GFP transgene andimmunohistochemically stained for endothelial marker CD144 (FAST REDChromogen (SIGNET) from Covance Research Products, Inc); CD144expression is confined to cells forming lumen-like spaces lined by cellsthat co-express GFP and CD144 (indicated by dual staining). FIGS. 2f andg . show low (f) and high (g) magnifications of immunohistochemistry forABCB5 protein; reactivity is restricted to anastomosing channelsidentical to those seen in panel c. The inset in panel f depicts similarformation of ABCB5-reactive channels in a patient-derived melanomabiopsy. FIG. 2h . depicts in situ hybridization for ABCB5 mRNA revealinga channel pattern corresponding to that of ABCB5 protein expression(compare with panel f; inset is sense control). FIG. 2i . shows thedetection of anti-ABCB5 mAb using anti-mouse Ig immunohistochemistry inmelanoma xenografts after intravenous administration in vivo; notelocalization to channels (inset represents anti-mouse Ig staining afterintravenous administration of irrelevant isotype-matched control mAb).FIG. 2j . shows dual-labeling immunofluorescence microscopy for ABCB5(left panel), CD144 (middle panel), and ABCB5 & CD144 (right panel).FIG. 2k shows dual-labeling immunofluorescence microscopy for ABCB5(left panel), TIE-1 (middle panel), and ABCB5 & TIE-1 (right panel).

FIGS. 3A-3F depict the interdependency of MMIC-driven vasculogenesis andtumourigenesis. FIG. 3a . shows representative flow cytometric ABCB5expression or control staining (FITC, F11) plotted against forwardscatter (FSC) for human A375, MUM-2B, and MUM-2C melanoma cell inocula.FIG. 3b . shows representative histologic sections of melanomas thatdeveloped from three unsegregated and ABCB5-depleted melanoma cell linesinjected intradermally into human skin xenografts. FIG. 3c . showshistologically determined tumour formation rate (%) 3 weeks followingintradermal transplantation of unsegregated vs. ABCB5⁺-depleted humanA375, MUM-2B or MUM-2C melanoma cells (2×10⁶/inoculum) into humanskin/Rag2^(−/−) chimeric mice (n=5, respectively). FIG. 2d . showshistological tumour volumes (mean±s.e.m.) 3 weeks following intradermaltransplantation of unsegregated vs. ABCB5⁺-depleted human A375, MUM-2Bor MUM-2C melanoma cells (2×10⁶/inoculum) into human skin/Rag2^(−/−)chimeric mice. FIG. 3e shows immunohistochemistry for laminin revealingextent of channel formation in melanomas that developed fromunsegregated or ABCB5⁺-depleted melanoma cell inocula derived from A375,MUM-2B or MUM-2C lines injected intradermally into human skin xenografts(arrows=necrosis). FIG. 3f depicts image analysis of lamininimmunoreactivity for melanomas derived from unsegregated andABCB5⁺-depleted cell inocula; y-axis is percent of pixelated area withreactivity (mean±s.e.m.); solid bar represents tumours derived fromunsegregated melanoma cells, open bars represent tumours derived fromABCB5⁺-depleted cells (A375, P<0.0032; MUM-2B, P<0.0005; MUM-2C,P<0.0059).

FIGS. 4A and 4B depict an analysis of the correlation of ABCB5 proteinand mRNA expression across human melanoma cell lines. FIG. 4a . showswestern blots of ABCB5 and tubulin expression of a panel of humanmelanoma cell lines. FIG. 4b shows relative ABCB5 mRNA expression (log2) in a panel of human melanoma cell lines plotted against ABCB5 proteinexpression as determined by ratios of ABCB5 89 kD western blot bandintensity and tubulin western blot band intensity for each humanmelanoma cell line. Data points in FIG. 4b are: 1, SK-MEL-2; 2,SK-MEL-5; 3, SK-MEL-28; 4, MDA-MB-435; 5, UACC-62; 6, UACC-257; 7, M14;8, MALME-3M. r, Spearman Rank Correlation r (corrected for ties).

DETAILED DESCRIPTION

The present invention relates in part to the discovery that numerousCSC-associated genes have altered expression or function in cancer stemcells, e.g., melanoma stem cells. In some aspects, the invention relatesto diagnostic arrays and methods for detecting cancer, e.g., melanoma,in an individual based on the expression of CSC-associated genes. Inother aspects, the invention relates to compositions, kits, and methodsuseful for treating individuals having cancer. In some embodiments, thetreatment methods involve modulating e.g., inducing or inhibiting, theactivity of CSC-associated genes. The CSC-associated genes can bemodulated by any one of a number of ways known in the art and describedherein e.g., overexpression, RNAi-based inhibition, etc. In some cases,the CSC-associated genes encode cell surface proteins which, whenupregulated in cancer stem cells, may be selectively targeted forisolating, e.g., by flow cytometry, identifying, e.g., byimmunolabeling, and killing of cancer stem cells, e.g., melanoma stemcells.

The mechanism by which CSCs cause tumor formation and growth and thepotential role of CSC-specific differentiation plasticity intumorigenicity are currently unknown. It has been demonstrated accordingto the invention that CSC play an important role in providingnutritional support to growing tumors. For instance we have shown herein(Examples) a selective capacity of ABCB5⁺ malignant melanoma initiatingcells (MMIC)³ to undergo vasculogenic differentiation and to generateblood-perfused vessel-like channels in vivo. A repertoire of genesdifferentially expressed in MMIC compared to tumour bulk populationswere identified by microarray analyses on purified ABCB5⁺ and ABCB5⁻cell subsets derived from the established human melanoma cell lines andfrom three separate patient-derived melanoma specimens. Using thisapproach, 399 genes were identified that were differentially expressedbetween ABCB5±MMIC and ABCB5⁻ melanoma bulk populations. The genes,which are outlined in Tables 1-8, are referred to herein asCSC-associated genes. Of the CSC-associated genes, 265 were upregulated(Table 1; Table 1 includes Table 1.1 and Table 1.2) and 150 weredownregulated (Table 2). For certain CSC-associated genes, subcellularlocation, e.g., plasma membrane, nucleus, etc., gene type, e.g., enzyme,complex, transporter, etc., and drugs that affect, e.g., target, theiractivity are identified (Table 3). A summary of those annotations andnetworks is provide in Table 3. Genes that function share a commonpathway have a common “network”) designation in Table 3. SomeCSC-associated genes, e.g., those which have “plasma membrane”annotations, encode proteins that are associated with the cell surface.Such cell surface proteins are useful in a variety ways. For example,cell surface proteins that are upregulated in cancer stem cells, may beselectively targeted, e.g., using the methods disclosed herein, forisolating, identifying, and killing of cancer stem cells. A listing ofexemplary cell surface proteins encoded by CSC-associated genes isprovided in Table 4.

TABLE 1.1 Upregulated CSC-associated genes (p < 0.05) GENESYMBOL ID FoldChange HECW1 237295_at 11.843 RP11-139H14.4 1569124_at 11.472 CDC16242359_at 6.261 ANK2 202921_s_at 4.162 LOC146325 1553826_a_at 3.943UGT1A6 206094_x_at 3.86 C12ORF51 1557529_at 3.632 SNRPA1 242146_at 3.54PDE4B 215671_at 3.457 PAPD4 222282_at 3.39 ZNF536 233890_at 3.303 KSR2230551_at 3.211 BUB1 233445_at 3.209 ZNF292 236435_at 3.201 CABIN11557581_x_at 3.052 SDAD1 242190_at 3.009 ASCC3L1 214982_at 3.009 ZNF224216983_s_at 2.986 KIDINS220 1557246_at 2.97 WIPF2 216006_at 2.916C12ORF51 230216_at 2.874 VPS37B 236889_at 2.85 NARG1 1556381_at 2.827LOC145757 1558649_at 2.779 SDCCAG8 243963_at 2.67 ZNF154 242170_at 2.667ZFR 238970_at 2.655 TRPV1 1556229_at 2.636 ANAPC5 235926_at 2.631 CUL4A232466_at 2.607 TRIO 240773_at 2.607 LOC283888 1559443_s_at 2.56RAB11FIP3 228613_at 2.546 PTK2 234211_at 2.539 MYO10 243159_x_at 2.528NAT8B 206964_at 2.513 CDC14B 234605_at 2.512 TRIM33 239716_at 2.496 SF1210172_at 2.452 SGCA 1562729_at 2.395 LOC285147 236166_at 2.377 N4BP2L2242576_x_at 2.349 HNRPH1 213472_at 2.332 FLJ10357 241627_x_at 2.31PHF20L1 219606_at 2.3 ANKRD28 241063_at 2.297 TRNT1 243236_at 2.295GOLGA8A 213650_at 2.289 KIAA1618 231956_at 2.27 RBM5 209936_at 2.249LOC645513 239556_at 2.24 LOC729397 236899_at 2.231 PABPN1 213046_at2.228 SVIL 215279_at 2.228 PIP5K3 1557719_at 2.227 STRAP 1558002_at2.189 KIAA2013 1555933_at 2.18 NUPL1 241425_at 2.179 IFNGR1 242903_at2.171 AKAP9 215483_at 2.168 LOC254128 1557059_at 2.164 IRS2 236338_at2.162 RHOA 240337_at 2.143 JARID2 232835_at 2.139 GPD2 243598_at 2.13RADIL 223693_s_at 2.126 CROP 242389_at 2.121 EXT1 242126_at 2.116 XRCC5232633_at 2.106 PDXDC1 1560014_s_at 2.105 MEF2C 236395_at 2.104 ZNF567242429_at 2.103 ZNF337 1565614_at 2.096 TTLL4 1557611_at 2.092 FUBP1240307_at 2.087 NPTN 228723_at 2.086 TPM4 235094_at 2.079 NCKAP1L209734_at 2.071 KRTAP19-1 1556410_a_at 2.07 SLC30A9 237051_at 2.063HDAC3 240482_at 2.062 C10ORF18 244165_at 2.046 SMA4 238446_at 2.035 GBF1233114_at 2.03 GABPA 243498_at 2.03 SFRS15 243759_at 2.028 CREB3L2237952_at 2.013 SLC2A8 239426_at 2.012 N4BP2L1 213375_s_at 2.01 IDS1559136_s_at 2.001 COBRA1 1556434_at 1.985 TXNL1 243664_at 1.98LOC388135 230475_at 1.979 MTUS1 239576_at 1.975 TAF15 227891_s_at 1.971HNRPD 241702_at 1.962 TRIM46 238147_at 1.96 NBR1 1568856_at 1.957 WDR68233782_at 1.924 HNRPD 235999_at 1.92 BLID 239672_at 1.91 LOC145786229178_at 1.907 HOXD3 206601_s_at 1.897 AOC3 204894_s_at 1.894 PRPF38B230270_at 1.888 SLC20A1 230494_at 1.884 SEC16B 1552880_at 1.877 FLT1232809_s_at 1.861 HUWE1 214673_s_at 1.858 BUB1 216277_at 1.856 GPR135241085_at 1.851 PSEN1 242875_at 1.851 KIAA0907 230028_at 1.83 POLR2J21552622_s_at 1.828 SFRS15 222311_s_at 1.818 CBS 240517_at 1.818 ETS1241435_at 1.797 LRRFIP1 239379_at 1.796 OCIAD1 235537_at 1.794 LRCH3229387_at 1.793 CCDC14 240884_at 1.771 HNRNPC 235500_at 1.769 DCUN1D2240478_at 1.76 NPAS2 1557690_x_at 1.76 POFUT2 207448_at 1.759 CHD2244443_at 1.757 TMEM165 1560622_at 1.756 FLJ31306 239432_at 1.753 HPS1239382_at 1.749 WTAP 1560274_at 1.747 TNPO1 1556116_s_at 1.739 ZFHX3215828_at 1.737 AKR1CL2 1559982_s_at 1.732 C20ORF4 234654_at 1.731CCDC57 214818_at 1.703 MALAT1 224568_x_at 1.699 EWSR1 229966_at 1.686MYO10 244350_at 1.677 MALAT1 223940_x_at 1.659 ATXN2L 207798_s_at 1.656PDK1 239798_at 1.654 POLR2J2 1552621_at 1.652 CENPJ 220885_s_at 1.64PDSS1 236298_at 1.64 UNK 1562434_at 1.637 BDP1 224227_s_at 1.632 N4BP2L2235547_at 1.631 MDM4 235589_s_at 1.629 SNORA28 241843_at 1.628 ZFX207920_x_at 1.625 NAPA 239362_at 1.624 PRO1073 228582_x_at 1.607 MLL212079_s_at 1.599 SGOL2 235425_at 1.591 RBM25 1557081_at 1.57 BARD1205345_at 1.559 LOC388969 232145_at 1.555 GGT1 211417_x_at 1.555 FAM62C239770_at 1.551 TTC9C 1569189_at 1.55 TCAG7.907 238678_at 1.546 OSGEP242930_at 1.541 RHOBTB2 1556645_s_at 1.538 C5ORF24 229098_s_at 1.531RBM4 213718_at 1.53 SLC2A11 232167_at 1.529 DDX17 213998_s_at 1.528C22ORF30 216555_at 1.521 C9ORF85 244160_at 1.52 DNM1L 236032_at 1.503SQLE 213577_at 1.502 CRIPAK 228318_s_at 1.486 ZNF800 227101_at 1.484RAD54L 204558_at 1.483 TAF1B 239046_at 1.468 THRAP3 217847_s_at 1.464CNIH3 232758_s_at 1.451 UQCC 229672_at 1.451 HOXA2 228642_at 1.44 RBM26229433_at 1.43 RFT1 240281_at 1.426 MTERFD3 225341_at 1.422 LOC641298208118_x_at 1.419 ZNF326 241720_at 1.418 NBPF16 201104_x_at 1.411 ASPM232238_at 1.411 RNF43 228826_at 1.401 IPW 213447_at 1.399 TTC3208664_s_at 1.396 USP36 224979_s_at 1.393 KIAA0841 36888_at 1.389 NEK1213328_at 1.381 AMZ2 227567_at 1.377 TBC1D8 204526_s_at 1.373 STK36231806_s_at 1.362 SF3B1 214305_s_at 1.359 HELLS 242890_at 1.359 SYNE2202761_s_at 1.356 KIAA1267 224489_at 1.355 C14ORF135 1563259_at 1.353SF3B1 201070_x_at 1.35 CLN8 229958_at 1.344 STK36 234005_x_at 1.335ZNF226 219603_s_at 1.332 COQ4 218328_at 1.328 DTX3 49051_g_at 1.32 WFS11555270_a_at 1.315 ZNF251 226754_at 1.313 ARS2 201679_at 1.307 ATAD2235266_at 1.304 CCDC73 239848_at 1.294 BCL9L 227616_at 1.291 MET213816_s_at 1.283 NFATC2IP 217527_s_at 1.272 CHD8 212571_at 1.27 TNRC6A234734_s_at 1.268 OSBPL5 233734_s_at 1.261 COIL 203653_s_at 1.259 CPEB2226939_at 1.251 TBC1D8 221592_at 1.246 RUNX3 204198_s_at 1.233 LBA1213261_at 1.225 CENPJ 234023_s_at 1.22 MARCH6 201737_s_at 1.219 ANKRD44226641_at 1.218 NAPE-PLD 242635_s_at 1.216 C12ORF48 220060_s_at 1.216CCDC93 219774_at 1.208 ZUFSP 228330_at 1.205 SMC6 218781_at 1.203 TAOK3220761_s_at 1.195 JARID1A 226367_at 1.192 DCLRE1C 242927_at 1.187 TTC26233999_s_at 1.184 EIF4G3 201935_s_at 1.174 ORMDL1 223187_s_at 1.171TCOF1 202385_s_at 1.169 CCDC52 234995_at 1.166 PMS2L3 214473_x_at 1.159HERC5 219863_at 1.156 CASC5 228323_at 1.144 SON 201085_s_at 1.144 APBB240148_at 1.139 LOC338799 226369_at 1.137 PHC1 218338_at 1.123 DEPDC1232278_s_at 1.119 NRP2 210841_s_at 1.106 ZMYND8 209049_s_at 1.102 CEP55218542_at 1.096

TABLE 1.2 Highly upregulated genes as detected by RT-PCR ABCB5+/ ABCB5−Description Gname Fold change Angiopoietin-like 3 ANGPT5 3.0596Brain-specific angiogenesis inhibitor FLJ41988 3.0596 1 Cadherin 5, type2, VE-cadherin 7B4/CD144 3.0596 (vascular epithelium) Epidermal growthfactor (beta- HOMG4/URG 187.8365 urogastrone) C-fos induced growthfactor VEGF-D/VEGFD 3.5884 (vascular endothelial growth factor D)Hepatocyte growth factor F-TCF/HGFB 4.542 (hepapoietin A; scatterfactor) Heparanase HPA/HPR1 286.6871 Insulin-like growth factor 1 IGFI4.7022 (somatomedin C) Jagged 1 (Alagille syndrome) AGS/AHD 1566.5046Laminin, alpha 5 KIAA1907 3.8727 Platelet/endothelial cell adhesionCD31/PECAM-1 11.9037 molecule (CD31 antigen) Plexin domain containing 1DKFZp686F0937/ 3.4184 TEM3 Stabilin 1 CLEVER-1/FEEL-1 4.357 Transforminggrowth factor, alpha TFGA 3549.3357 Tumor necrosis factor (TNFDIF/TNF-alpha 4.0652 superfamily, member 2) Vascular endothelial growthfactor C Flt4-L/VRP 446.7529

TABLE 2 Downregulated CSC-associated genes (p < 0.05) GENESYMBOL ID FoldChange ECHDC1 233124_s_at 0.943 DARS 201624_at 0.928 GALNT1 201722_s_at0.926 CGGBP1 224600_at 0.913 CSE1L 201112_s_at 0.911 GMFB 202544_at0.904 RPL7L1 225515_s_at 0.899 SKP1 200718_s_at 0.898 IGHMBP2215980_s_at 0.893 LOC137886 212934_at 0.886 CSE1L 210766_s_at 0.885ERRFI1 224657_at 0.881 MAP2K4 203266_s_at 0.881 TNFAIP1 201207_at 0.88TBXA2R 207554_x_at 0.877 SEPHS1 208940_at 0.875 IPO7 200993_at 0.875C16ORF63 225087_at 0.872 INSIG2 209566_at 0.872 TFB1M 228075_x_at 0.87PAK1 226507_at 0.869 C14ORF156 221434_s_at 0.867 SMYD2 212922_s_at 0.867ENTPD5 231676_s_at 0.867 PPP3CA 202457_s_at 0.867 MBNL1 201152_s_at0.867 MRPL42 217919_s_at 0.866 SUPT7L 201838_s_at 0.865 PMP22210139_s_at 0.865 GABARAPL2 209046_s_at 0.863 PITPNA 201190_s_at 0.863C2ORF30 224630_at 0.851 TXNDC12 223017_at 0.849 POP4 202868_s_at 0.847MRPL51 224334_s_at 0.846 AK3 224655_at 0.845 GPR107 211979_at 0.843TMEM126B 221622_s_at 0.843 PSMA2 201316_at 0.839 KIAA1737 225623_at0.837 TRAPPC2L 218354_at 0.837 RLBP1L1 224996_at 0.835 CCDC127 226515_at0.835 CPNE3 202119_s_at 0.833 HIAT1 225222_at 0.832 MECR 218664_at 0.832ACBD6 225317_at 0.83 SLC16A1 202235_at 0.83 ANXA4 201302_at 0.83 DNAJC21230893_at 0.829 C22ORF28 200042_at 0.829 SPOPL 225659_at 0.828 PDHB211023_at 0.827 EIF2S1 201144_s_at 0.824 LOC645166 228158_at 0.823CAMK2D 225019_at 0.823 LIMS1 212687_at 0.822 VTI1B 209452_s_at 0.821 YY1224711_at 0.821 TRAPPC2 219351_at 0.821 LOC126917 225615_at 0.819 STX8204690_at 0.819 NANP 228073_at 0.817 NDFIP1 217800_s_at 0.815 UBE3C1560739_a_at 0.815 KPNA6 226976_at 0.814 C19ORF42 219097_x_at 0.813DHX40 218277_s_at 0.812 NUCB2 203675_at 0.812 RAB1A 213440_at 0.81 USP8229501_s_at 0.808 MAP1LC3B 208785_s_at 0.808 PDHB 208911_s_at 0.807SH2B3 203320_at 0.806 PPP1R3D 204554_at 0.805 DEGS1 209250_at 0.804HSDL2 209513_s_at 0.803 LOC203547 225556_at 0.802 CANX 238034_at 0.8PSMA3 201532_at 0.798 PIGY 224660_at 0.793 CYB5R3 1554574_a_at 0.793BRI3 223376_s_at 0.792 CREB1 204313_s_at 0.791 LOC389203 225014_at 0.79WDR41 218055_s_at 0.789 C9ORF78 218116_at 0.789 GNPDA1 202382_s_at 0.787RPE 225039_at 0.787 HSPA4L 205543_at 0.786 SEPT11 201307_at 0.784 HEATR2241352_at 0.784 ENAH 222433_at 0.783 MED19 226300_at 0.782 TBC1D5201814_at 0.782 EMP2 225079_at 0.781 STX11 235670_at 0.778 ANKH229176_at 0.776 ENDOD1 212573_at 0.775 IL13RA1 201887_at 0.775 RAB14200927_s_at 0.772 TMEM30A 232591_s_at 0.771 DDX52 212834_at 0.771 PTPMT1229535_at 0.769 SRPRB 218140_x_at 0.767 FAM98A 212333_at 0.767 SRP72208803_s_at 0.766 RPE 221770_at 0.766 HOXB9 216417_x_at 0.766 MAEA207922_s_at 0.765 GHITM 1554510_s_at 0.764 CAPZB 201949_x_at 0.764ANKRD52 228257_at 0.762 MOBKL1B 214812_s_at 0.762 MIA3 1569057_s_at0.759 UBE2E3 210024_s_at 0.758 CAMK2D 228555_at 0.758 UBXD7 212840_at0.754 C18ORF10 213617_s_at 0.754 HSD17B1 228595_at 0.753 PDLIM5212412_at 0.752 SRP72 208801_at 0.751 ZNF618 226590_at 0.75 TSPAN31203227_s_at 0.744 MAP3K15 200979_at 0.741 C18ORF10 212055_at 0.737 ATP5I207335_x_at 0.737 TOX4 201685_s_at 0.73 TBXA2R 336_at 0.73 COL4A2211966_at 0.729 TIMM23 218119_at 0.723 NDUFAF2 228355_s_at 0.722 FOXN3218031_s_at 0.721 EIF2S1 201142_at 0.717 NDUFB6 203613_s_at 0.712 TM6SF11558102_at 0.704 ELOVL2 213712_at 0.699 PPP1R7 201213_at 0.698 BAT3230513_at 0.697 ZNF668 219047_s_at 0.691 ERBB3 1563253_s_at 0.691C12ORF45 226349_at 0.688 PGRMC2 213227_at 0.686 NUDT4 212183_at 0.685AABHD7 239579_at 0.661 CEP27 228744_at 0.651 RAB11FIP3 216043_x_at 0.551FHL3 218818_at 0.546 NAALAD2 1554506_x_at 0.464 LOC219731 1557208_at0.419

TABLE 3 CSC-genes annotations Entrez Gene ID for Fold Name HumanAffymetrix Change Networks Location Type Drugs Actin — 1 Unknown groupADA 100 — 8 Cytoplasm enzyme pentostatin, vidarabine Adaptor protein 2 —8 Unknown complex AFP 174 — 5 Extracellular transporter Space AGT 183 —8 Extracellular other Space AHR 196 — 7 Nucleus ligand- dependentnuclear receptor AKAP9 10142 215483_at 2.168 1 Cytoplasm other Akt — 2Unknown group amino acids — 6 Unknown chemical - endogenous mammalianAMPH 273 — 8 Plasma other Membrane AMZ2 51321 227567_at 1.377 8 Unknownother ANAPC1 64682 — 4 Nucleus other ANAPC10 10393 — 4 Nucleus enzymeANAPC11 51529 — 4 Unknown enzyme ANAPC13 25847 — 4 Unknown other ANAPC229882 — 4 Nucleus enzyme ANAPC4 29945 — 4 Unknown enzyme ANAPC5 51433235926_at 2.631 4 Nucleus enzyme ANAPC7 51434 — 4 Unknown other ANK2 287202921_s_at 4.162 4 Plasma other Membrane ANKRD28 23243 241063_at 2.29713 Unknown other AOC3 8639 204894_s_at 1.894 2 Plasma enzyme MembraneAP2A2 161 — 8 Cytoplasm transporter APBB2 323 40148_at 1.139 9 Cytoplasmother APP 351 — 9 Plasma other AAB-001 Membrane ARD1A 8260 — 8 Nucleusenzyme Arf — 8 Unknown group ARF5 381 — 8 Cytoplasm transporter ARHGDIB397 — 7 Cytoplasm other ASCC3L1 23020 214982_at 3.009 9 Nucleus enzyme(includes EG: 23020) ASCL1 429 — 9 Nucleus transcription regulator ASPM259266 232238_at 1.411 3 Nucleus other ATAD2 29028 235266_at 1.304 7Unknown other ATP — 9 Unknown chemical - endogenous mammalian ATXN2L11273 207798_s_at 1.656 9 Unknown other BARD1 580 205345_at 1.559 1Nucleus transcription regulator BCL2 596 — 6 Cytoplasm other oblimersen,(−)-gossypol BCL9L 283149 227616_at 1.291 6 Cytoplasm other BDP1 55814224227_s_at 1.632 9 Nucleus transcription regulator beta-estradiol — 3Unknown chemical - endogenous mammalian BRF1 2972 — 9 Nucleustranscription regulator BUB1 (includes 699 233445_at 3.209 5 Nucleuskinase EG: 699) BUB1B 701 — 4 Nucleus kinase C12ORF48 55010 220060_s_at1.216 Unknown other C12ORF51 283450 1557529_at 3.632 4 Unknown otherCABIN1 23523 1557581_x_at 3.052 1 Nucleus other Calmodulin — 1 Unknowngroup CASC5 57082 228323_at 1.144 3 Nucleus other CASP3 836 — 4Cytoplasm peptidase IDN-6556 CASP6 839 — 9 Cytoplasm peptidase CBS 875240517_at 1.818 1 Cytoplasm enzyme CD151 977 — 7 Plasma other MembraneCDC14B 8555 234605_at 2.512 5 Nucleus phosphatase CDC16 8881 242359_at6.261 4 Nucleus other CDC20 991 — 3 Nucleus other CDC23 (includes 8697 —4 Nucleus enzyme EG: 8697) CDC26 246184 — 4 Nucleus other CDC27 996 — 4Nucleus other CDC5L 988 222179_at 1.292 9 Nucleus other CDK2 1017 — 7Nucleus kinase BMS-387032, flavopiridol CDKN1A 1026 — 7 Nucleus kinaseCDT1 81620 — 7 Nucleus other CDX1 1044 — 9 Nucleus transcriptionregulator CENPJ 55835 220885_s_at 1.64 4 Nucleus transcription regulatorCEP55 55165 218542_at 1.096 5 Unknown other CHD8 57680 212571_at 1.27 1Nucleus enzyme CHEK2 11200 — 5 Nucleus kinase CHRM3 1131 — 8 PlasmaG-protein fesoterodine, Membrane coupled ABT-089, receptoratropine/edrophonium, cyclopentolate/ phenylephrine,ipratropium/albuterol, trihexyphenidyl, carbamylcholine, darifenacin,methacholine, diphenhydramine, quinidine, procyclidine, trospium,atropine sulfate/benzoic acid/hyoscyamine/ methenamine/methyleneblue/phenyl salicylate, homatropine, dicyclomine, methantheline,orphenadrine, fluoxetine/olanzapine, doxacurium, aspirin/caffeine/orphenadrine, propantheline, tridihexethyl, biperiden, anisotropinemethylbromide, glycopyrrolate, diphenhydramine/8- chlorotheophylline,atropine/ hyoscyamine/ phenobarbital/ scopolamine, atropinesulfate/diphenoxylate hydrochloride, pipecuronium, flavoxate,chlorpheniramine/ methscopolamine/ phenylephrine, mepenzolic acid,atropine sulfate/difenoxin hydrochloride, homatropine methylbromide,hydroxyamphetamine/ tropicamide, cisatracurium, hyoscyamine/phenobarbital, bethanechol, olanzapine, oxybutynin, tropicamide,solifenacin, cyclopentolate, tolterodine, cevimeline, acetylcholine,ipratropium, atropine, pilocarpine, benztropine, hyoscyamine, arecoline,scopolamine, N- methylscopolamine, tiotropium, carbinoxamine, buclizine,diphenhydramine/ phenylephrine, brompheniramine CIB1 10519 — 7 Nucleusother Ck2 — 1 Unknown complex CKM 1158 — 5 Cytoplasm kinase CLIC1 1192 —9 Nucleus ion channel CLIC4 25932 — 6 Cytoplasm ion channel CLIC5 53405— 1 Cytoplasm ion channel CLN8 2055 229958_at 1.344 Cytoplasm other COIL8161 203653_s_at 1.259 6 Nucleus other COL4A1 1282 — 5 Extracellularother collagenase Space COPB1 1315 — 8 Cytoplasm transporter Creb — 2Unknown group CREB3L2 64764 237952_at 2.013 3 Unknown other CRIPAK285464 228318_s_at 1.486 Cytoplasm other CROP 51747 242389_at 2.121 7Nucleus other CRY1 1407 — 7 Nucleus enzyme CTNNA1 1495 — 6 Plasma otherMembrane CTNNAL1 8727 — 6 Plasma other Membrane CTNNB1 1499 — 6 Nucleustranscription regulator CUL4A 8451 232466_at 2.607 1 Nucleus other DAPK11612 — 6 Cytoplasm kinase DCLRE1C 64421 242927_at 1.187 Nucleus enzymeDDX17 10521 213998_s_at 1.528 6 Nucleus enzyme DENND4A 10260 230607_at2.368 1 Nucleus other DMD 1756 — 8 Plasma other Membrane DNM1L 10059236032_at 1.503 2 Cytoplasm enzyme DSN1 79980 — 3 Nucleus other DTX — 3Unknown group DTX1 1840 — 3 Nucleus transcription regulator DTX2 113878— 3 Nucleus other DTX3 196403 49051_g_at 1.32 3 Cytoplasm other DUB — 18Unknown group DVL1 1855 — 6 Cytoplasm other DVL2 1856 — 6 Cytoplasmother Dynamin — 2 Unknown group EGFR 1956 — 7 Plasma kinase cetuximab,AEE Membrane 788, panitumumab, BMS-599626, ARRY-334543, XL647,canertinib, gefitinib, HKI-272, PD 153035, lapatinib, vandetanib,erlotinib EIF4G3 8672 201935_s_at 1.174 4 Cytoplasm translationregulator EPOR 2057 — 9 Plasma transmembrane erythropoietin, Membranereceptor darbepoetin alfa, continuous erythropoietin receptor activatorERBB2 2064 — 3 Plasma kinase trastuzumab, BMS- Membrane 599626, ARRY-334543, XL647, CP- 724, 714, HKI-272, lapatinib, erlotinib ETS1 2113241435_at 1.797 2 Nucleus transcription regulator EWSR1 2130 229966_at1.686 1 Nucleus other EXT1 2131 242126_at 2.116 4 Cytoplasm enzyme FLOT110211 — 3 Plasma other Membrane FLT1 2321 232809_s_at 1.861 2 Plasmakinase sunitinib, axitinib, Membrane CEP 7055 FMR1 2332 — 7 Nucleusother FRK 2444 — 9 Nucleus kinase FUBP1 8880 240307_at 2.087 1 Nucleustranscription regulator FZR1 51343 — 4 Nucleus other GABPA 2551243498_at 2.03 2 Nucleus transcription regulator GBF1 8729 233114_at2.03 8 Cytoplasm other GGT1 2678 211417_x_at 1.555 6 Cytoplasm enzymeGPD2 2820 243598_at 2.13 3 Cytoplasm enzyme HDAC3 8841 240482_at 2.062 1Nucleus transcription tributyrin, PXD101, regulator pyroxamide,MGCD0103, vorinostat, FR 901228 HECW1 23072 237295_at 11.843 6 Cytoplasmenzyme HELLS 3070 242890_at 1.359 3 Nucleus enzyme HERC5 51191 219863_at1.156 6 Cytoplasm enzyme Histone h3 — 1 Unknown group HNRNPC 3183235500_at 1.769 1 Nucleus other HNRPD 3184 241702_at 1.962 4 Nucleustranscription regulator HNRPH1 3187 213472_at 2.332 8 Nucleus otherHOXA2 3199 228642_at 1.44 8 Nucleus transcription regulator HOXD3 3232206601_s_at 1.897 7 Nucleus transcription regulator HPS1 3257 239382_at1.749 14 Cytoplasm other HPS4 89781 — 14 Cytoplasm other HSPA5 3309 — 3Cytoplasm other HUWE1 10075 214673_s_at 1.858 6 Nucleus transcriptionregulator IFNG 3458 — 9 Extracellular cytokine Space IFNGR1 3459242903_at 2.171 4 Plasma transmembrane interferon Membrane receptorgamma-1b IL1B 3553 — 4 Extracellular cytokine IL-1 trap Space Insulin —2 Unknown group IRS2 8660 236338_at 2.162 2 Cytoplasm other ITGB3 3690 —7 Plasma transmembrane TP 9201, Membrane receptor EMD121974, tirofibanITPR1 3708 — 4 Cytoplasm ion channel JARID1A 5927 226367_at 1.192 9Nucleus transcription regulator JARID2 3720 232835_at 2.139 4 Nucleustranscription regulator Jnk — 2 Unknown group KIAA1267 284058 224489_at1.355 1 Nucleus other KIDINS220 57498 1557246_at 2.97 6 Nucleustranscription regulator KIR2DL3 3804 — 9 Plasma other Membrane KITLG(includes 4254 — 9 Extracellular growth factor EG: 4254) Space KLF6 1316— 5, 9 Nucleus transcription regulator LCN2 3934 — 9 Extracellulartransporter Space LMO2 4005 — 9 Nucleus other LOC388135 388135 230475_at1.979 5 Unknown other LRRFIP1 9208 239379_at 1.796 3 Nucleustranscription regulator MALAT1 378938 224568_x_at 1.699 Unknown otherMapk — 2 Unknown group MEF2C 4208 236395_at 2.104 2 Nucleustranscription regulator MET 4233 213816_s_at 1.283 2 Plasma kinaseMembrane mGluR — 8 Unknown group MIS12 79003 — 3 Nucleus other MLL 4297212079_s_at 1.599 1 Nucleus transcription regulator MPL 4352 — 9 Plasmatransmembrane SB-497115 Membrane receptor MTUS1 57509 239576_at 1.975 1Unknown other MYC 4609 — 6 Nucleus transcription regulator MYF6 4618 — 5Nucleus transcription regulator MYO10 4651 243159_x_at 2.528 3 Cytoplasmother MYOD1 4654 — 5 Nucleus transcription regulator N4BP2L1 90634213375_s_at 2.01 Unknown other Nap125 — 16 Unknown group NAPA 8775239362_at 1.624 2 Cytoplasm transporter NAPE-PLD 222236 242635_s_at1.216 8 Cytoplasm enzyme NARG1 80155 1556381_at 2.827 8 Nucleustranscription regulator NAT13 80218 — 8 Cytoplasm enzyme NBPF15 284565201104_x_at 1.411 1 Unknown other NBR1 4077 1568856_at 1.957 5 Unknownother NCKAP1L 3071 209734_at 2.071 16 Plasma other Membrane NCOA3 8202 —7 Nucleus transcription regulator NEK1 4750 213328_at 1.381 6 Nucleuskinase NES 10763 — 5 Cytoplasm other NFATC2IP 84901 217527_s_at 1.272 1Nucleus other NFkB — 2 Unknown complex NFKBIE (includes 4794 — 13Nucleus transcription EG: 4794) regulator NMB 4828 — 8 Extracellularother Space NPAS2 4862 1557690_x_at 1.76 7 Nucleus transcriptionregulator NPTN 27020 228723_at 2.086 1 Plasma other Membrane NRP2 8828210841_s_at 1.106 2, 3 Plasma kinase Membrane NUPL1 9818 241425_at 2.17917 Nucleus transporter OGG1 4968 — 9 Nucleus enzyme OSBPL5 114879233734_s_at 1.261 3 Cytoplasm other OSGEP 55644 242930_at 1.541 3Unknown peptidase P38 MAPK — 2 Unknown group PABPN1 8106 213046_at 2.2285 Nucleus other PAX3 5077 — 7 Nucleus transcription regulator PCBP1(includes 5093 — 6 Nucleus translation EG: 5093) regulator PDE4B 5142215671_at 3.457 2 Cytoplasm enzyme dyphylline, nitroglycerin,arofylline, tetomilast, L 869298, aminophylline, anagrelide, cilomilast,milrinone, rolipram, dipyridamole, L- 826, 141, roflumilast,tolbutamide, theophylline, pentoxifylline, caffeine PDE5A 8654 239556_at2.24 4 Cytoplasm enzyme dyphylline, nitroglycerin, DA- 8159,aminophylline, sildenafil, dipyridamole, aspirin/dipyridamole,vardenafil, tolbutamide, tadalafil, theophylline, pentoxifylline PDGF BB— 2 Unknown complex PDK1 5163 239798_at 1.654 1 Cytoplasm kinase PDSS123590 236298_at 1.64 15 Unknown enzyme PDXDC1 23042 1560014_s_at 2.105 8Unknown other PHC1 1911 218338_at 1.123 5 Nucleus other PI3K — 2 Unknowncomplex PIP5K1C 23396 — 7 Plasma kinase Membrane PIP5K3 2005761557719_at 2.227 2 Cytoplasm kinase Pka — 1 Unknown complex Pkc(s) — 2Unknown group PLAA 9373 — 4 Cytoplasm other PLC gamma — 2 Unknown groupPld — 8 Unknown group PLK1 5347 — 7 Nucleus kinase BI 2536 PMS2L3 5387214473_x_at 1.159 3 Unknown other POLR2J2 246721 1552622_s_at 1.828 1Nucleus transcription regulator POU4F2 5458 — 6 Nucleus transcriptionregulator PP2A — 6 Unknown complex PRDM5 11107 — 5 Nucleus other PRKCB15579 — 7 Cytoplasm kinase enzastaurin, ruboxistaurin progesterone — 8Unknown chemical - endogenous mammalian PSEN1 5663 242875_at 1.851 2Plasma peptidase (R)-flurbiprofen Membrane PTEN 5728 — 3 Cytoplasmphosphatase PTK2 5747 234211_at 2.539 2 Cytoplasm kinase PTPN12 5782 — 7Cytoplasm phosphatase PTPN14 5784 — 6 Cytoplasm phosphatase PTPRA 5786 —7 Plasma phosphatase Membrane PTPRD 5789 — 6 Plasma phosphatase MembranePTPRE 5791 — 7 Plasma phosphatase Membrane PTPRS (includes 58021556116_s_at 1.739 7 Plasma phosphatase EG: 5802) Membrane RAB11FIP39727 228613_at 2.546 8 Cytoplasm other RAB11FIP4 84440 — 8 Cytoplasmother Rac — 2 Unknown group RAD50 10111 — 5 Nucleus enzyme RAD54L 8438204558_at 1.483 5 Nucleus enzyme RB1 5925 — 9 Nucleus transcriptionregulator RBM25 58517 1557081_at 1.57 7 Nucleus other RBM4 5936213718_at 1.53 7 Nucleus other RBM5 10181 209936_at 2.249 6 Nucleusother RDBP 7936 — 3 Nucleus other RHOA 387 240337_at 2.143 2 Cytoplasmenzyme RHOBTB2 23221 1556645_s_at 1.538 Unknown enzyme RNA polymerase II— 1 Unknown complex RNU1A 6060 — 1 Unknown other RP13-122B23.3 259201556434_at 1.985 3 Nucleus other RPL10 6134 — 6 Cytoplasm other RUNX3864 204198_s_at 1.233 7 Nucleus transcription regulator SBF1 6305 — 3Plasma phosphatase Membrane SCMH1 22955 — 5 Nucleus transcriptionregulator SCN3A 6328 — 4 Plasma ion channel riluzole Membrane SEC16A9919 — 10 Cytoplasm phosphatase SEC16B 89866 1552880_at 1.877 10 Nucleusother Secretase gamma — 9 Unknown complex SF1 7536 210172_at 2.452 1, 4Nucleus transcription regulator SF3B1 23451 214305_s_at 1.359 1 Nucleusother SFRS15 57466 243759_at 2.028 Nucleus other SGCA 6442 1562729_at2.395 8 Plasma other Membrane SGCB 6443 — 8 Plasma other Membrane SGCD6444 — 8 Cytoplasm other SGCG 6445 — 8 Plasma other Membrane SH2D1A(includes 4068 — 5 Cytoplasm other EG: 4068) SKIL 6498 — 4 Nucleustranscription regulator SLC29A1 2030 — 9 Plasma transporter MembraneSLC2A11 66035 232167_at 1.529 9 Plasma other Membrane SLC2A8 29988239426_at 2.012 Plasma transporter Membrane SLC30A9 10463 237051_at2.063 7 Nucleus transporter SLC4A1 6521 — 9 Plasma transporter MembraneSMAD4 4089 — 6 Nucleus transcription regulator SMARCA5 8467 — 9 Nucleustranscription regulator SMC5 23137 — 12 Nucleus other SMC6 79677218781_at 1.203 12 Nucleus other SMN1 6606 — 6 Nucleus other SNRPA1 6627242146_at 3.54 8 Nucleus other SNW1 22938 — 5 Nucleus transcriptionregulator SON 6651 201085_s_at 1.144 5 Nucleus other SP4 6671 — 3Nucleus transcription regulator sphingomyelin — 9 Unknown chemical -endogenous mammalian SPN 6693 — 3 Plasma transmembran Membrane ereceptor SPTBN1 6711 — 4, 6, 8 Plasma other Membrane SQLE 6713 213577_at1.502 3 Cytoplasm enzyme SQSTM1 8878 — 5 Cytoplasm transcriptionregulator SRC 6714 — 6 Cytoplasm kinase dasatinib, AZM-475271 STK3627148 231806_s_at 1.362 6 Unknown kinase STRAP 11171 1558002_at 2.189 2Plasma other Membrane STX3 6809 — 7 Plasma transporter Membrane SUMO17341 — 8 Nucleus enzyme SUMO2 6613 — 9 Nucleus other SVIL 6840 215279_at2.228 4 Plasma other Membrane SYNE2 23224 202761_s_at 1.356 1 Nucleusother TAF15 8148 227891_s_at 1.971 1 Nucleus transcription regulatorTAF1A 9015 — 5 Nucleus transcription regulator TAF1B 9014 239046_at1.468 5 Nucleus transcription regulator TAF1C 9013 — 5 Nucleustranscription regulator TAOK3 51347 220761_s_at 1.195 2 Cytoplasm kinaseTap — 17 Unknown complex TBC1D8 11138 204526_s_at 1.373 3 Plasma otherMembrane TCERG1 10915 — 8 Nucleus transcription regulator TCF7L2 6934 —6 Nucleus transcription regulator TCOF1 (includes 6949 202385_s_at 1.1691 Nucleus transporter EG: 6949) TCR — 2 Unknown complex TERF2 7014 — 5Nucleus other TH1L 51497 — 3 Nucleus other THAP7 80764 — 1 Nucleus otherTHRAP3 9967 217847_s_at 1.464 1 Nucleus transcription regulator TIMP17076 — 9 Extracellular other Space TNF 7124 — 4 Extracellular cytokineadalimumab, Space etanercept, infliximab, CDP870, golimumab, thalidomideTNRC6A 27327 234734_s_at 1.268 9 Nucleus other TP53 7157 — 5 Nucleustranscription regulator TP53BP1 7158 — 5 Nucleus transcription regulatorTPM4 7171 235094_at 2.079 8 Cytoplasm other Trans- — 15 Unknown grouphexaprenyl- transtransferase TRIM33 51592 239716_at 2.496 6 Nucleustranscription regulator TRIO 7204 240773_at 2.607 2 Plasma kinaseMembrane tRNA — 19 Unknown group adenylyl- transferase tRNA — 19 Unknowngroup cytidylyl- transferase TRNT1 51095 243236_at 2.295 19 Cytoplasmenzyme TRPV1 7442 1556229_at 2.636 2 Plasma ion channel SB-705498,Membrane capsaicin TSG101 7251 — 5, 7 Nucleus transcription regulatorTSPAN7 7102 — 7 Plasma other Membrane TTC3 7267 208664_s_at 1.396 7Cytoplasm other TXNL1 9352 243664_at 1.98 9 Cytoplasm enzyme Ubiquitin —1 Unknown group UGT — 7 Unknown group UGT1A6 54578 206094_x_at 3.86 7Cytoplasm enzyme USP36 57602 224979_s_at 1.393 18 Nucleus peptidase Vegf— 2 Unknown group VEGFA 7422 — 3 Extracellular growth factorbevacizumab, Space ranibizumab, pegaptanib VEGFB (includes 7423 — 3Extracellular growth factor EG: 7423) Space VPS28 51160 — 5 Cytoplasmtransporter VPS37B 79720 236889_at 2.85 5 Nucleus other WAS 7454 — 11Cytoplasm other WDR68 10238 233782_at 1.924 4 Cytoplasm other WFS1 74661555270_a_at 1.315 3 Cytoplasm enzyme WIPF2 147179 216006_at 2.916 11Unknown other WT1 7490 — 6 Nucleus transcription regulator WTAP 95891560274_at 1.747 2 Nucleus other XRCC5 7520 232633_at 2.106 5 Nucleusenzyme YWHAG 7532 — 4 Cytoplasm other ZEB1 6935 — 5 Nucleustranscription regulator ZFHX3 463 215828_at 1.737 5 Nucleustranscription regulator ZFR 51663 238970_at 2.655 Nucleus other ZFX 7543207920_x_at 1.625 9 Nucleus transcription regulator ZMYND8 23613209049_s_at 1.102 7 Nucleus transcription regulator ZNF224 7767216983_s_at 2.986 6 Nucleus other ZNF226 7769 219603_s_at 1.332 8Nucleus transcription regulator ZNF326 284695 241720_at 1.418 Nucleustranscription regulator ZNF536 9745 233890_at 3.303 Unknown other ZWINT(includes 11130 — 3 Nucleus other EG: 11130)

TABLE 4 Cell Surface Genes Entrez Gene ID for Human Name Location 7204TRIO Plasma Membrane 1956 EGFR Plasma Membrane 7102 TSPAN7 PlasmaMembrane 977 CD151 Plasma Membrane 2064 ERBB2 Plasma Membrane 2321 FLT1Plasma Membrane 2030 SLC29A1 Plasma Membrane 11171 STRAP Plasma Membrane8828 NRP2 Plasma Membrane 4233 MET Plasma Membrane 273 AMPH PlasmaMembrane 351 APP Plasma Membrane 1756 DMD Plasma Membrane 1495 CTNNA1Plasma Membrane 8727 CTNNAL1 Plasma Membrane 10211 FLOT1 Plasma Membrane3459 IFNGR1 Plasma Membrane 23396 PIP5K1C Plasma Membrane 5663 PSEN1Plasma Membrane 6445 SGCG Plasma Membrane 6693 SPN Plasma Membrane 6711SPTBN1 Plasma Membrane 6840 SVIL Plasma Membrane 2057 EPOR PlasmaMembrane 5789 PTPRD Plasma Membrane 4352 MPL Plasma Membrane 5786 PTPRAPlasma Membrane 27020 NPTN Plasma Membrane 3690 ITGB3 Plasma Membrane7442 TRPV1 Plasma Membrane 8639 AOC3 Plasma Membrane 1131 CHRM3 PlasmaMembrane 3804 KIR2DL3 Plasma Membrane 287 ANK2 Plasma Membrane 3071NCKAP1L Plasma Membrane 5791 PTPRE Plasma Membrane 5802 PTPRS PlasmaMembrane 6305 SBF1 Plasma Membrane 6328 SCN3A Plasma Membrane 6442 SGCAPlasma Membrane 6443 SGCB Plasma Membrane 66035 SLC2A11 Plasma Membrane29988 SLC2A8 Plasma Membrane 6521 SLC4A1 Plasma Membrane 6809 STX3Plasma Membrane 11138 TBC1D8 Plasma Membrane

A used herein “CSC-associated gene” refers to a gene whose expression orfunction is altered in cancer stem cells. CSC-associated genes includegenes whose expression is signficantly altered, e.g., significantlyupregulated or significantly downregulated, in cancer stem cellscompared with non-cancer stem cells, e.g., cancer cells that are notstem cells, normal cells, etc. In some embodiments, genes that havesignificantly altered expression levels in cancer stem cells areidentified by using an appropriate statistical test for establishing thesignificance of differences between expression levels in a cancer stemcell and a non-cancer stem cell. Tests for statistical significance arewell known in the art and are exemplified in Applied Statistics forEngineers and Scientists by Petruccelli, Chen and Nandram 1999 ReprintEd. The magnitude of up-, or down-, regulated expression of aCSC-associated gene in a cancer stem cell compared with a non-cancerstem cell may vary. In some embodiments, the expression level of aCSC-associated gene is at least 10%, at least 25%, at least 50%, atleast 100%, at least 250%, at least 500%, or at least 1000% higher, orlower, than its expression level in a non-cancer stem cell. In otherembodiments, the expression level of a CSC-associated gene is at least2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least50-fold, at least 100-fold, or more higher, or lower, than itsexpression level a non-cancer stem cell.

CSC-associated genes are not limited to genes which are upregulated ordownregulated in cancer stem cells. In some embodiments, aCSC-associated gene is a gene that may or may not have alteredexpression in a cancer stem cell, but that nevertheless functions in apathway that is deregulated in cancer stem cells. Typically, deregulatedpathways in cancer stem cells involve the product(s) of one or moregenes whose expression is upregulated or downregulated and/or theproduct(s) of one or more genes with altered functionality, e.g., due toa mutation, thereby resulting in altered function of the pathway, e.g.,overactivity or underactivity of the pathway.

In some embodiments, CSC-associated genes are identified in cancer stemcells of a breast cancer, prostate cancer, colon cancer, lung cancer,renal cancer or melanoma. In some instances, CSC-associated genes areidentified in cancer stem cells of a melanoma, which are also referredto as malignant melanoma initiating cells (MMIC). Other cancer stemcells (e.g., non-ABCB5 CSCs) are known in the art.

Exemplary CSC-associated genes are disclosed in Tables 1-8. In someembodiments, a CSC-associated gene is selected from the group consistingof: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11,SLC2A8, SLC4A1, STX3, and TBC1D8. In some embodiments, theCSC-associated gene is one that is not a gene of the group consisting ofEGFR, CD151, ERBB2, FLT1, SLC29A1, NRP2, MET, AMPH, APP, DMD, and ITGB3.In some embodiments, the CSC-associated gene is one that is not a geneof the group consisting of: TRIO, TSPAN7, STRAP, CTNNA1, CTNNAL1, FLOT1,IFNGR1, PIP5K1C, PSEN1, SGCG, SPN, SPTBN1, SVIL, EPOR, PTPRD, MPL,PTPRA, NPTN, TRPV1, AOC3, CHRM3, and KIR2DL3. In some embodiments, theCSC-associated gene is one that is not a gene that has previously beenindicated as a tumor suppressor or oncogene. In some embodiments, theCSC-associated gene is one that is not a gene of the group consistingof: EWSR1, TP53, EGFR, ITPR1, NBR1, MLL, PTK2, PTPN14, RB1, JARID1A,SKIL, TNF, TP53BP1, TRIO, SF1, TAF15, NCOA3, RAD54L, CUL4A, SMARCA5,RAD50, AKAP9, DENND4A, DDX17, HECW1, ZMYND8, ANAPC13, ANAPC5, TH1L,TRIM33, and CHD8. In some embodiments, the CSC-associated gene is onethat is not a gene of the group consisting of: BARD1, BCL2, CBS, CTNNB1,ERBB2, EWSR1, HPS1, IFNG, IL1B, PTEN, TP53, VEGFA, CHEK2, and HPS4.

In part, the disclosure relates to CSC-associated genes as well as theRNAs and polypeptides (CSC-associated RNA and polypeptides) that theyencode and antibodies and antigen-binding fragments that specificallybind them. The CSC-associated genes, RNAs and polypeptides, encompassvariants, homologues, and fragments. Variants may result fromalternative splicing or allelic variation of certain genes provided inTables 5. In general, homologues and alleles typically will share atleast 90% nucleotide identity and/or at least 95% amino acid identity tothe sequences of the cancer antigen nucleic acids and polypeptides,respectively, in some instances will share at least 95% nucleotideidentity and/or at least 97% amino acid identity, in other instanceswill share at least 97% nucleotide identity and/or at least 98% aminoacid identity, in other instances will share at least 99% nucleotideidentity and/or at least 99% amino acid identity, and in other instanceswill share at least 99.5% nucleotide identity and/or at least 99.5%amino acid identity. Homology can be calculated using various, publiclyavailable software tools known in the art, such as those developed byNCBI (Bethesda, Md.) that are available through the internet. Exemplarytools include the BLAST system (e.g., using the default nucleic acid(Blastn) or protein (Blastp) search parameters) available from thewebsite of the National Center for Biotechnology Information (NCBI) atthe National Institutes of Health.

The CSC-associated genes are, among other things, useful for diagnosingcancer, such as breast cancer, prostate cancer, colon cancer, lungcancer, renal cancer or melanoma. Because CSC-associated gene expressionis altered in cancer (e.g., upregulated or downregulated), theexpression level of CSC-associated gene(s), e.g., a gene listed in Table5 or 7, in an individual is diagnostic of cancer in that individual.Accordingly, the diagnostic methods disclosed herein can involvedetermining the CSC-associated RNA or protein (polypeptide) levels.

The term “individual” as used herein means any mammalian individual orsubject, including, e.g., humans and non-human mammals, such asprimates, rodents, and dogs. Individuals specifically intended fordiagnosis and treatment using the methods described herein arepreferably humans.

The expression level of CSC-associated gene(s) may be determined byusing any of a number of methods known in the art. In some embodiments,the expression levels are determined from a biological sample (e.g., atest sample) obtained from a individual (e.g., a human). Exemplary,biological samples include an isolated cell, an isolated tissue, saliva,gingival secretions, cerebrospinal fluid (spinal fluid),gastrointestinal fluid, mucus, urogenital secretions, synovial fluid,blood, serum, plasma, urine, cystic fluid, lymph fluid, ascites, pleuraleffusion, interstitial fluid, intracellular fluid, ocular fluids,seminal fluid, mammary secretions, vitreal fluid, and nasal secretions.However, biological samples are not so limited and other exemplarybiological specimens will be readily apparent to one of ordinary skillin the art. For the purposes of diagnosing melanoma, for example, thebiological sample is preferably a skin tissue sample, e.g., a skinbiopsy containing a suspicious lesion.

Expression levels of CSC-associated genes may be determined fordiagnostic purposes using nucleic acid hybridization or nucleic acidamplification to detect the mRNAs that they encode. Methods for nucleicacid hybridization or amplification are well known in the art. In someembodiments, the nucleic acid amplification is real-time RT-PCR orRT-PCR. Other methods known to one of ordinary skill in the art could beemployed to analyze mRNA levels, for example nucleic acid arrays, cDNAanalysis, Northern analysis, and RNase Protection Assays. Nucleic acidarrays may be used to assay (e.g., for diagnostic purposes) theexpression levels of multiple CSC-associated genes in parallel. Othersuitable nucleic acid detection methods will be apparent to the skilledartisan.

Expression levels of CSC-associated genes may be determined fordiagnostic purposes by detecting the polypeptides that they encode.Methods for detecting polypeptides are well known in the art. Exemplarypolypeptide detection methods include, but are not limited to, EnzymeLinked Immunosorbent Assays (ELISA), radioimmunoassays (RIA), sandwichimmunometric assays, flow cytometry, western blot assays,immunoprecipitation assays, immunohistochemistry, immunomicroscopy,lateral flow immuno-chromatographic assays, BIACORE technology, andproteomics methods, such as mass spectroscopy. Antibody arrays may beused to assay (e.g., for diagnostic purposes) the expression levels ofmultiple CSC-associated genes in parallel. Other suitable polypeptidedetection methods will be apparent to the skilled artisan.

In some embodiments, e.g., where polypeptide, antibody or nucleic acidarrays are used, expression levels of up to 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156,157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170,171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268,269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324,325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352,353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380,381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394,395, 396, 397, 398, 399, or more CSC-associated genes may be tested inparallel.

The diagnostic methods of the invention involve a comparison betweenexpression levels of CSC-associated genes in a test sample and areference value. The results of the comparison are diagnostic of cancer,e.g., melanoma. In some embodiments, the reference value is theexpression level of the gene in a reference sample. A reference valuemay be a predetermined value and may also be determined from referencesamples (e.g., control biological samples) tested in parallel with thetest samples. A reference value may be a positive or negative controllevel. A reference value can be a single cut-off value, such as a medianor mean or a range of values, such as a confidence interval. Referencevalues can be established for various subgroups of individuals, such asindividuals predisposed to cancer, individuals having early or latestage cancer, male and/or female individuals, or individuals undergoingcancer therapy. The level of the reference value will depend upon theparticular population or subgroup selected. For example, an apparentlyhealthy population will have a different “normal” value than will apopulation which has cancer, or a population that has a predispositionfor cancer. Appropriate ranges and categories for reference values canbe selected with no more than routine experimentation by those ofordinary skill in the art.

The reference sample can be any of a variety of biological samplesagainst which a diagnostic assessment may be made. Examples of referencesamples include biological samples from control populations or controlsamples. Reference samples may be generated through manufacture to besupplied for testing in parallel with the test samples, e.g., referencesample may be supplied in diagnostic kits. When the reference sample isfrom a cancer, e.g., tumor tissue, the expression level of the referencesample (the reference value) is the expression level of theCSC-associated gene in the cancer. Similarly, when the reference sampleis a normal sample, e.g., non-tumor tissue, the expression level of thereference sample (the reference value) is the expression level of theCSC-associated gene in the non-tumor tissue. Similarly, when thereference sample is a cancer stem cell sample, the expression level ofthe reference sample (the reference value) is the expression level ofthe CSC-associated gene in the cancer stem cell sample. In someembodiments, the reference sample is of a melanoma and the expressionlevel of the reference sample is the expression level of theCSC-associated gene in melanoma. In some embodiments, the referencesample is of a non-melanoma tissue and the expression level of thereference sample is the expression level of the CSC-associated gene innon-melanoma tissue. Other appropriate reference samples will beapparent to the skilled artisan.

The diagnostic methods are based in part on a comparison of expressionlevels of CSC-associated genes between test samples and referencesample. In some embodiments, if the expression level of theCSC-associated gene in the test sample is about equal to the expressionlevel of the CSC-associated gene in the reference sample, then the testsample and reference sample are likely of a similar origin, category orclass. For example, if expression levels in a test sample and referencesample are about the same (e.g., not statistically significantlydifferent), and the reference sample is from a normal tissue, then thetest sample is likely a normal tissue sample, and a normal diagnosiscould be indicated. Alternatively, if expression levels in a test sampleand reference sample are about the same, and the reference sample isfrom a cancer tissue, then the test sample is likely a cancer sample,and a diagnosis of cancer could be indicated. In certain embodiments, ifthe expression level in a test sample and reference sample are about thesame, and the reference sample is from a melanoma, then the test sampleis likely a melanoma sample, and a diagnosis of melanoma could beindicated.

In some cases, depending on factors such as the particularCSC-associated gene(s) being evaluated, the condition being diagnosed,and the type of reference sample, an expression level of aCSC-associated gene in a test sample that is statistically significantlyhigher or statistically significantly lower than its expression level ina reference sample indicates a diagnosis. For example, when theCSC-associated gene is among those listed in Table 1 or 8 and thereference value is the expression level of the CSC-associated gene in anormal (e.g., non-cancerous) reference sample, if the expression levelof the CSC-associated gene in the test sample is significantly higherthan the expression level of the CSC-associated gene in the normalreference sample, the comparison indicates cancer, e.g., melanoma.Similarly, when the CSC-associated gene is among those listed in Table 1or 8 and the reference value is the expression level of theCSC-associated gene in a cancer, e.g., melanoma, reference sample, ifthe expression level of the CSC-associated gene in the test sample issignificantly lower than the expression level of the CSC-associated genein the cancer reference sample, the comparison does not indicate cancer.Alternatively, when the CSC-associated gene is among those listed inTable 2 or 7 and the reference value is the expression level of theCSC-associated gene in a normal reference sample, if the expressionlevel of the CSC-associated gene in the test sample is significantlylower than the expression level of the CSC-associated gene in the normalreference sample, the comparison indicates cancer. Similarly, when theCSC-associated gene is in Table 2 or 7 and the reference value is theexpression level of the CSC-associated gene in a cancer, e.g., melanoma,reference sample, if the expression level of the CSC-associated gene inthe test sample is significantly higher than the expression level of theCSC-associated gene in the cancer reference sample, the comparison doesnot indicate melanoma. Appropriate combinations of particularCSC-associated gene(s), conditions to be diagnosed, and types ofreference samples, can be selected with no more than routineexperimentation by those of ordinary skill in the art for use in thediagnostic methods disclosed herein.

The magnitude of the difference between the test sample and referencesample that is sufficient to indicate a diagnosis will depend on avariety of factors such as the particular CSC-associated gene(s) beingevaluated, the condition being diagnosed, heterogeneity in healthy ordisease populations from which samples are drawn, the type of referencesample, the magnitude of expression level of a CSC-associated gene, theassay being used, etc. It is well within the purview of the skilledartisan to determine the appropriate magnitude of difference between thetest sample and reference sample that is sufficient to indicate adiagnosis. In some embodiments, the expression level of theCSC-associated gene in the test sample is at least 10%, at least 20%, atleast 50%, at least 100%, at least 200%, at least 500%, at least 1000%or more higher than the expression level of the gene in the referencesample. In other embodiments, the expression level of the CSC-associatedgene in the test sample is at least 10%, at least 20%, at least 50%, atleast 100%, at least 200%, at least 500%, at least 1000% or more lowerthan the expression level of the gene in the reference sample.

Some CSC-associated genes that are normally produced in very lowquantities but whose production is dramatically increased in tumorcells, e.g., a CSC-associated gene in Table 1 or 8, can trigger animmune response. Thus, in some instances, specific immunoreactivityagainst a CSC-associated polypeptide, e.g., a polypeptide encoded by agene listed in Table 1 or 8, in a individual may be diagnostic of cancerin the individual. Immunoreactivity against CSC-associated polypeptidesmay be humoral or cellular, and is associated with a specific immuneresponse to a CSC-associated polypeptide that is upregulated in a cancerstem cell in an individual. In the case of a humoral response thediagnostic methods may involve detecting the presence of one or moreantibodies in an individual that specifically bind CSC-associatedpolypeptides. Methods for detecting antibodies are disclosed herein(e.g., ELISA, peptide arrays, etc.) and are well known in the art. Insome cases, the presence of CSC-polypeptide specific effector cells isdiagnostic of an immune response specific to that CSC-polypeptide.

T Lymphocytes recognize complexes of peptide ligands (e.g.,CSC-associated polypeptides) and Major Histocompatibility Complex (MHC)molecules presented at the surface of Antigen Presenting Cells (APC).Class I tetramers bind to a distinct set of T cell receptors (TCRs) on asubset of CD8+ T cells, and Class II tetramers bind to a distinctpopulation of CD4+ T cells. Methods for detecting antigen-specific Tcells using MHC tetramers are well known in the art (e.g., NewMicroarray Detects Antigen-Specific T Cells and Immune Responses. PLoSBiol 1(3): e76 2003) and can be used to detect CSC-polypeptide specificT cells which may be diagnostic of cancer in an individual. ITAGreagents, for example, from Beckman Coulter provide a convenient way tomeasure the cellular response directed toward a single CSC-associatedpeptide using MHC tetramers.

The methods for evaluating expression of CSC-associated genes, e.g.,diagnostic methods, disclosed herein may be combined with methods fortreating an individual having or suspected of having cancer. Thetreatment may comprise a step of determining the expression level of theCSC-associated gene in the individual. The treatment may also comprise astep of comparing the expression level of the CSC-associated gene to areference value, such that the results of the comparison are diagnosticof cancer in the individual. In certain cases, if the comparison resultsin a diagnosis of cancer in the individual, the administering step isperformed. In some cases, after a diagnosis is made using the methodsdisclosed herein, a treatment plan is selected. For example, if adiagnostic assay reveals that the expression of a particularCSC-associated gene is altered, e.g., increased or decreased, comparedto a normal reference sample, then a treatment directed at thatparticular CSC-associated gene may be implemented. The diagnosticmethods can also be used to evaluate the response to a treatment. Forexample, the determining and the comparing may be repeated at one ormore intervals after the administering step to evaluate the response tothe treatment.

CSC-associated polypeptide arrays and arrays of antibodies that bindCSC-associated polypeptides may be constructed by immobilizing largenumbers of isolated CSC associated polypeptides or antibodies, orantigen binding fragments, to a solid support. Methods for producingpolypeptide and antibody arrays are well known in the art. The methodstypically involve production of proteins (CSC-associated polypeptides orantibodies) from an expression library, cloned into E. coli, yeast, ormammalian cells, or similar system, e.g., hybridomas etc., from whichthe expressed proteins are then purified, for example via His, GST tag,or Protein A/G affinity purification. Cell free proteintranscription/translation is an alternative for synthesis of proteinswhich do not express well in bacterial or other in vivo systems. Thepurified isolated CSC associated polypeptides or antibodies areimmobilized on the array surface (solid support surface) using art knownmethods. For example, proteins can be immobilized by adsorption,covalent (e.g., aldehydes) and non-covalent (e.g., biotin-streptavidin)interactions. Other methods of conjugation will be readily apparent toone of ordinary skill in the art. In some embodiments, the polypeptidearrays of the invention consist essentially of at least 2, at least 5,at least 10, at least 20, at least 50, at least 100, or morepolypeptides or immunogenic fragments thereof encoded by aCSC-associated gene set forth in Table 1, 5, 7, or 8. In someembodiments, the antibody arrays of the invention consist essentially ofat least 2, at least 5, at least 10, at least 20, at least 50, at least100, or more different antibodies or antigen-binding fragments thatspecifically bind polypeptides (CSC-associated polypeptides) encoded bya CSC-associated gene set forth in Table 1, 5, 7, or 8.

Methods for producing nucleic acid arrays are well known in the art.Nucleic acid arrays may be constructed by, e.g., immobilizing to a solidsupport large numbers oligonucleotides, polynucleotides, or cDNAs havingsequences complementary to CSC-associated mRNAs. The skilled artisan isalso referred to Chapter 22 “Nucleic Acid Arrays” of Current ProtocolsIn Molecular Biology (Eds. Ausubel et al. John Wiley and #38; Sons NY,2000), International Publication WO00/58516, U.S. Pat. Nos. 5,677,195and 5,445,934 which provide exemplary methods relating to nucleic acidarray construction and use in detection of nucleic acids of interest. Insome embodiments, the nucleic acid arrays consist essentially of atleast 2, at least 5, at least 10, at least 20, at least 50, at least100, at least 200, at least 300, or more of the CSC-associated genes setforth in Table 1, 5, 7, or 8.

In some embodiments, the expression levels of multiple CSC-associatedgenes may be combined to produce an expression profile. As used herein,“expression profile” refers to a set of expression levels of a plurality(e.g., 2 or more) of CSC-associated genes. Expression profiles have avariety of uses. For example, expression profiles may be used toclassify (or sub-classify) a sample, preferably a clinical sample. Insome embodiments, reference samples, for which a classification, e.g., adisease category, e.g., breast cancer, prostate cancer, melanoma, etc.,has already been ascertained, are used to produce known expressionprofiles. In some embodiments, the similarity of an expression profileof a test sample and a known expression profile, is assessed bycomparing the level of the same CSC-associated gene in the test andknown expression profiles (i.e., expression level pairs). In some cases,a test expression profile is compared with one or more members of aplurality of known expression profiles, and a known expression profilethat most closely resembles (i.e., is most similar to) the test profileis identified. In certain cases, the classification of a knownexpression profile that is identified as similar to a test expressionprofile is assigned to the test expression profile, thereby classifyingthe clinical sample associated with the test expression profile. Themethods are useful for classifying samples across a range of phenotypes,e.g., disease status, risk status, etc., based on expression profiles.In some embodiments, a classification model (e.g., discriminantfunction, naïve bayes, support vector machine, logistic regression, andothers known in the art) may be built based on the reference expressionprofiles from various samples from individuals known to have differentdisorders (e.g., cancers) and/or from healthy individuals, and used toclassify subsequently obtained samples (e.g., clinical samples).

The invention also provides methods for stratifying a populationcomprising individuals having cancer. In some embodiments, methodsinvolve determining expression levels of at least 2, at least 5, atleast 10, at least 20, at least 50, at least 100, at least 200, at least300, or more of the CSC-associated genes set forth in Table 5, 7, or 8,for example, by using the arrays of the invention, and stratifying thepopulation based on the expression levels. The stratification methodsare useful in epidemiological studies, for example, to identifysubpopulations of individuals at risk of cancer. The methods are alsouseful in clinical studies to identify patient subpopulations thatrespond better or worse to a particular treatment.

In some aspects, CSC-associated genes provide a basis for identifying,isolating, cloning, propagating, and expanding CSC populations in vitro.The present invention contemplates any suitable method of employingagents, e.g., isolated peptides, e.g., antibodies, that bind toCSC-associated polypeptides to separate CSCs from other cells.Accordingly, included in the present invention is a method of producinga population of isolated CSCs. The methods involve contacting a sample,e.g., a cell suspension, with one or a combination of agents, e.g.,antibodies or antigen binding fragments or ligands, which recognize andbind to an epitope, e.g., a cell surface protein, includingCSC-associated polypeptides (e.g., polypeptides encoded by the geneslisted in Table 4), on the CSC and separating and recovering from thesample the cells bound by the agents. The CSC-associated polypeptide maybe encoded by a CSC-associated gene that is selected from the groupconsisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB,SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.

In some instances, commerically available antibodies or antibodyfragments that bind CSC-associated polypeptides may be used in themethods disclosed herein. For example, antibodies against ANK2 include,e.g., rabbit anti-human Ankyrin brain polyclonal antibody from Abcam;mouse anti-human ANK2 monoclonal antibody clone 2.2 from Genway Biotech,Inc.; and mouse anti-human Ankyrin, brain variant 2 (ANK2) monoclonal,clone 2.20 and rabbit anti-human ankyrin, brain variant 2 (ank2)polyclonal from Lifespan Biosciences. Antibodies against NCKAP1Linclude, e.g., rabbit anti-human HEM1 polyclonal antibody fromProteintech Group, Inc. and are described in Weiner O D, et al., (2006)Hem-1 Complexes Are Essential for Rac Activation, Actin Polymerization,and Myosin Regulation during Neutrophil Chemotaxis. PLoS Biol 4(2): e38.Antibodies against PTPRE include, e.g., rabbit anti-PTPepsilon C-termRB0551-0552 polyclonal antibody from Abgent, mouse anti-human PTPREpolyclonal antibody from Abnova Corporation; mouse anti-PTPRE monoclonalantibody Clone 2D10 from Abnova Corporation; chicken anti-human PTPREpolyclonal antibody from Thermo Scientific; and Rabbit Anti-ProteinTyrosine phosphatase epsilon (PTPRE) antibody from Acris AntibodiesGmbH. Antibodies against PTPRS include, e.g., mouse anti-PTPRSmonoclonal antibody from Abcam; mouse anti-human PTPRS monoclonalantibody Clone 1H6 from Abnova Corporation; chicken anti-human PTPRSpolyclonal antibody from ABR-Affinity Bioreagents, now sold as ThermoScientific; chicken anti-human PTPRS polyclonal antibody from GeneTex;and mouse anti-human PTPRS monoclonal antibody, Clone 1H6 from GeneTex.Antibodies against SCN3A include, e.g., rabbit anti-SCN3A polyclonalantibody from Abcam; mouse anti-human SCN3A monoclonal antibody Clone3F3 from Abnova Corporation; and mouse Anti-human SCN3A monoclonalantibody Clone 3F3 from GeneTex. Antibodies against SCNB include, e.g.,mouse anti-beta Sarcoglycan Monoclonal Antibody from Abcam; mouseanti-human SGCB monoclonal antibody Clone 1C10 from Abnova Corporation;mouse anti-human SGCB monoclonal antibody Clone 1C10 from GeneTex; andrabbit anti-human Beta-sarcoglycan (SGCB) Polyclonal from LifespanBiosciences. Antibodies against SLC2A8 include, e.g., rabbit anti-humanGlucose Transporter 8 Polyclonal Antibody from Abcam; goatanti-GLUT8/SLC2A8 polyclonal antibody from Everest Biotech; rabbitanti-GLUCOSE TRANSPORTER 8 Polyclonal Antibody from GenWay Biotech,Inc.; rabbit Anti-Human GLUCOSE TRANSPORTER 5, C Terminus PolyclonalAntibody from GenWay Biotech, Inc.; goat anti-SLC2A8 polyclonal antibodyfrom IMGENEX; and rabbit anti-Human Solute Carrier Family 2 (FacilitatedGlucose Transporter) Member 8 (Slc2a8) polyclonal from LifespanBiosciences. Antibodies against SBF1 include, e.g., rabbit Anti-MTMRSC-term RB0717 polyclonal antibody from Abgent. Antibodies against SGCAinclude, e.g., mouse Anti-SGCA monoclonal antibody Clone 3C4 from AbnovaCorporation; rabbit Anti-Human SGCA polyclonal antibody from AtlasAntibodies; mouse Anti-SGCA monoclonal antibody clone 3C4 from NovusBiologicals; rabbit Anti-Human SGCA PRESTIGE ANTIBODIES fromSigma-Aldrich. Antibodies against SLC2A11 include, e.g., rabbitanti-human Solute Carrier Family 2 (Facilitated Glucose Transporter),Member 11 (Slc2a11) polyclonal from Lifespan Biosciences. Antibodiesagainst SLC4A1 include, e.g., rabbit anti-human Band 3, N Terminuspolyclonal antibody from Abcam; mouse anti-human SLC4A1 MaxPab®polyclonal antibody from Abnova Corporation; rabbit anti-human SLC4A1polyclonal antibody from Aviva Systems Biology; mouse anti-Band 3Monoclonal Antibody Clone BIII 136 from GenWay Biotech, Inc.; and mouseanti-human Solute Carrier Family 4, Anion Exchanger, Member 1 (SLC4A1)monoclonal Clone 3 h3 from Lifespan Biosciences. Antibodies against STX3include, e.g., rabbit anti-human STX3 Polyclonal Antibody from AtlasAntibodies and rabbit anti-human STX3 from Sigma-Aldrich. Antibodiesagainst TBC1D8 include, e.g., mouse anti-human TBC1D8 monoclonalantibody Clone 1A12 from Abnova Corporation; mouse anti-human TBC1D8monoclonal antibody Clone 1A12 from GeneTex; mouse anti-human TBC1D8monoclonal antibody Clone SS-18 from Santa Cruz Biotechnology, Inc.; andrabbit anti-human TBC1D8, aa 132-231 polyclonal antibody from StrategicDiagnostics, Inc.

Agents may be linked to a solid-phase and utilized to capture CSCs froma sample. The bound cells may then be separated from the solid phase byknown methods depending on the nature of the agent, e.g., antibody, andsolid phase. Alternatively, the agents may be conjugated to a detectablelabel, e.g., a fluorophore, that can be utilized to separate cells in aliquid phase, such as by fluorescent activated cell sorting. Exemplaryfluorophores are well known in the art (e.g., Invitrogen's MOLECULARPROBES technologies) and include FITC, TRITC, Cy3, Cy5, AlexaFluorescent Dyes, and Quantum Dots.

Systems appropriate for preparing the desired cell population includemagnetic bead/paramagnetic particle column utilizing isolated peptidesthat bind CSC-associated polypeptides for either positive or negativeselection; separation based on biotin or streptavidin affinity; and highspeed flow cytometric sorting of immunofluorescent-stained CSCs mixed ina suspension of other cells. Thus, the methods of the present inventioninclude the isolation of a population of CSCs.

Isolated CSCs may be prepared as substantially pure preparations. Theterm “substantially pure” means that a preparation is substantially freeof other cells. For example, an isolated CSC should constitute at least70 percent of the total cells present with greater percentages, e.g., atleast 85, 90, 95 or 99 percent, being preferred. The cells may bepackaged in a finished container such as a cryovial along with any othercomponents that may be desired, e.g., agents for preserving cells, orreducing bacterial growth. The CSCs are useful for a variety ofpurposes. The isolated cells may be used in basic research setting andin screening assays to identify compounds or compositions that affectgrowth of CSCs.

Isolated CSCs, prepared according to the methods disclosed herein, maybe useful in a drug discovery context for lead compound identificationand optimization in cell-based screens. For example, the effect of acompound on the growth and/or survival of a CSC may be determined in acell-based screen that uses an assay selected from: a cell countingassay, a replication labeling assay, a cell membrane integrity assay, acellular ATP-based viability assay, a mitochondrial reductase activityassay, a caspase activity assay, an Annexin V staining assay, a DNAcontent assay, a DNA degradation assay, and a nuclear fragmentationassay. Other exemplary assays include BrdU, EdU, or H3-Thymidineincorporation assays; DNA content assays using a nucleic acid dye, suchas Hoechst Dye, DAPI, Actinomycin D, 7-aminoactinomycin D or PropidiumIodide; Cellular metabolism assays such as AlamarBlue, MTT, XTT, andCellTitre Glo; Nuclear Fragmentation Assays; Cytoplasmic HistoneAssociated DNA Fragmentation Assay; PARP Cleavage Assay; TUNEL staining;and Annexin staining.

In some aspects, the agents for isolating CSCs are antibody orantigen-binding fragments. The antibodies and antigen binding fragmentsof the invention include monoclonal antibodies, polyclonal antibodies,human antibodies, chimeric antibodies, humanized antibodies,single-chain antibodies, F(ab′)₂, Fab, Fd, Fv, or single-chain Fvfragments.

Other aspects of the invention relate to treatment methods. In someembodiments, the methods involve modulating, e.g., inducing orinhibiting, the activity of CSC-associated genes (RNA or protein) and,thereby, inhibiting the growth and survival of cancer stem cells. Insome embodiments, the treatment methods involve selective delivery,e.g., by antibodies or antigen-binding fragments, of therapeutic agentsto cancer stem cells. The methods are useful for inhibiting theproliferation and/or survival of cancer stem cells and, therefore, areuseful for treating cancer, e.g., melanoma. The level of modulation,e.g., inducing or inhibiting, of the activity of a CSC-associated genethat is sufficient to affect the growth and/or survival of a cancer stemcell compared with a control level depends on a variety of factors,including the particular CSC-associated gene being modulated, the cancerstem cell within which the modulation occurs, and the level ofexpression in the control sample. It is well within the purview of theskilled artisan to determine the appropriate level of modulation that issufficient to sufficient to inhibit the growth and/or survival of acancer stem cell.

The term “inhibiting” refers to any decrease in expression level oractivity. As used herein, “inhibit”, “suppress”, or “reduce” may, or maynot, be complete. For example, cell proliferation, may, or may not, bedecreased to a state of complete arrest for an effect to be consideredone of suppression, inhibition or reduction of cell proliferation.Moreover, “suppress”, “inhibit”, or “reduce” may comprise themaintenance of an existing state and the process of effecting a changein state. For example, inhibition of cell proliferation may refer to theprevention of proliferation of a non-proliferating cell (maintenance ofa non-proliferating state) and the process of inhibiting theproliferation of a proliferating cell (process of affecting aproliferation state change). Similarly, inhibition of cell survival mayrefer to killing of a cell, or cells, such as by necrosis or apoptosis,and the process of rendering a cell susceptible to death, such as byinhibiting the expression or activity of an anti-apoptotic regulatoryfactor. The suppression, inhibition, or reduction may be at least about5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95%, 99% of control level (e.g., an untreatedstate). In some cases, the level of modulation (e.g., suppression,inhibition, or reduction) compared with a control level is statisticallysignificant. “Statistically signficant” is a term well known in the artand, for example, may refer to a p-value of less than 0.05, e.g., ap-value of less than 0.025 or a p-value of less than 0.01, using anappropriate statistical test (e.g., ANOVA, MANOVA, t-test, multiplecomparison test, etc.).

The methods involve treating an individual having, or at risk of having,cancer. As used herein an “individual at risk of having cancer” is anindividual, e.g., a human, with an increased likelihood of having cancercompared with a control population, e.g., a general population. Any oneof a number of risk factors known in the art may be evaluated todetermine whether or not an individual is at risk of having cancer. Forexample, factors that render an individual at risk of having melanomainclude, for example, UV exposure, family history of melanoma, personalhistory of melanoma, fair skin, freckles, high numbers of nevi (moles),light hair, age, gender, and Xeroderma pigmento sum.

The treatment methods disclosed herein may involve administering atherapeutically effective amount of a composition that induces theexpression of a CSC-associated gene which is downregulated in cancer(e.g., a gene in Table 2 or 7). In some instances, the composition thatinduces expression of a CSC-associated gene comprises a vector, such asan isolated plasmid, that expresses the CSC-associated gene.

As used herein, a “vector” may be any of a number of nucleic acidmolecules into which a desired sequence may be inserted by restrictionand ligation for transport between different genetic environments or forexpression in a host cell. Vectors are typically composed of DNAalthough RNA vectors are also available. Vectors include, but are notlimited to, plasmids, phagemids and virus genomes or portions thereof.

An expression vector is one into which a desired sequence may beinserted, e.g., by restriction and ligation such that it is operablyjoined to regulatory sequences and may be expressed as an RNAtranscript. Vectors may further contain one or more marker sequencessuitable for use in the identification of cells that have or have notbeen transformed or transfected with the vector. Markers include, forexample, genes encoding proteins that increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes thatencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and genesthat visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein).

Methods for identifying and obtaining coding sequences for use in themethods disclosed herein are routine in the art. For example, theskilled artisan may search Entrez Gene database using a GeneID orGeneAlias of a CSC-associated gene, e.g., a GeneID listed in Table 5, 7or 8, to identify transcripts associated with CSC-associated genes. Inmost cases, links to commercial suppliers (e.g., Open Biosystems) ofcDNA's containing the transcripts are provided in the Entrez Genewebinterface, which can be utilized to procure a copy cDNA clone. Inother cases, commerical sources (e.g., Sigma Aldrich) can be contacteddirectly.

As used herein, a coding sequence and regulatory sequences are said tobe “operably” joined when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide. It will be appreciated that a codingsequence need not encode a protein but may instead, for example, encodea functional RNA such as an shRNA.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Such 5′ non-transcribed regulatory sequences will include apromoter region that includes a promoter sequence for transcriptionalcontrol of the operably joined gene. Regulatory sequences may alsoinclude enhancer sequences or upstream activator sequences as desired.The vectors of the invention may optionally include 5′ leader or signalsequences. The choice and design of an appropriate vector is within theability and discretion of one of ordinary skill in the art. One of skillin the art will be aware of appropriate regulatory sequences forexpression of interfering RNA, e.g., shRNA, miRNA, etc.

In some embodiments, a virus vector for delivering a nucleic acidmolecule, an isolated plasmid, is selected from the group consisting ofadenoviruses, adeno-associated viruses, poxviruses including vacciniaviruses and attenuated poxviruses, Semliki Forest virus, Venezuelanequine encephalitis virus, retroviruses, Sindbis virus, and Tyvirus-like particle. Examples of viruses and virus-like particles whichhave been used to deliver exogenous nucleic acids include:replication-defective adenoviruses (e.g., Xiang et al., Virology219:220-227, 1996; Eloit et al., J. Virol. 7:5375-5381, 1997;Chengalvala et al., Vaccine 15:335-339, 1997), a modified retrovirus(Townsend et al., J. Virol. 71:3365-3374, 1997), a nonreplicatingretrovirus (Irwin et al., J. Virol. 68:5036-5044, 1994), a replicationdefective Semliki Forest virus (Zhao et al., Proc. Natl. Acad. Sci. USA92:3009-3013, 1995), canarypox virus and highly attenuated vacciniavirus derivative (Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353,1996), non-replicative vaccinia virus (Moss, Proc. Natl. Acad. Sci. USA93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.Stand. 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis etal., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et al.,Virology 212:587-594, 1995), lentiviral vectors (Naldini L, et al., ProcNatl Acad Sci USA. 1996 Oct. 15; 93(21):11382-8) and Ty virus-likeparticle (Allsopp et al., Eur. J. Immunol 26:1951-1959, 1996).

Another virus useful for certain applications is the adeno-associatedvirus, a double-stranded DNA virus. The adeno-associated virus iscapable of infecting a wide range of cell types and species and can beengineered to be replication-deficient. It further has advantages, suchas heat and lipid solvent stability, high transduction frequencies incells of diverse lineages, including hematopoietic cells, and lack ofsuperinfection inhibition thus allowing multiple series oftransductions. The adeno-associated virus can integrate into humancellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

Other useful viral vectors are based on non-cytopathic eukaryoticviruses in which non-essential genes have been replaced with the gene ofinterest. Non-cytopathic viruses include certain retroviruses, the lifecycle of which involves reverse transcription of genomic viral RNA intoDNA with subsequent proviral integration into host cellular DNA. Ingeneral, the retroviruses are replication-deficient (i.e., capable ofdirecting synthesis of the desired transcripts, but incapable ofmanufacturing an infectious particle). Such genetically alteredretroviral expression vectors have general utility for thehigh-efficiency transduction of genes in vivo. Standard protocols forproducing replication-deficient retroviruses (including the steps ofincorporation of exogenous genetic material into a plasmid, transfectionof a packaging cell lined with plasmid, production of recombinantretroviruses by the packaging cell line, collection of viral particlesfrom tissue culture media, and infection of the target cells with viralparticles) are provided in Kriegler, M., “Gene Transfer and Expression,A Laboratory Manual,” W.H. Freeman Co., New York (1990) and Murry, E. J.Ed. “Methods in Molecular Biology,” vol. 7, Humana Press, Inc., Clifton,N.J. (1991).

In some embodiments, isolated plasmid vectors comprises atumor-specific, e.g., melanoma-specific, e.g., a tyrosinase promoter,operably linked to the CSC-associated gene (See, e.g., Lillehammer, T.et al., Cancer Gene Therapy (2005) 12, 864-872). Other exemplarytumor-specific promoters are known in the art and will be apparent tothe skilled artisan.

The treatment methods may involve administering a therapeuticallyeffective amount of a composition that targets a product of aCSC-associated gene (i.e., Table 1), which are CSC-associated genes thatupregulated in cancer stem cells. The composition may target a productof a CSC-associated gene selected from the group set forth in Table 4,which are upregulated in cancer stem cells and are associated with thecell surface.

The product, e.g., mRNA or protein, of a CSC-associated gene can betargeted by any one of a number of methods known in the art. Forexample, the composition may comprise a gene knockdown reagent, e.g.,siRNA, that is complementary to a CSC-associated mRNA and inhibits itsexpression. In other embodiments, the composition may comprise anisolated molecule, e.g., antibody or antigen binding fragment, that isconjugated to a siRNA and that specifically binds to a CSC-associatedpolypeptide. Such antibody conjugated siRNAs (or similar genesuppression agents) may target a CSC-associated mRNA such as any ofthose encoded by the genes set forth in Table 1.

The CSC-associated gene may be selected from the following group ANK2,NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1,STX3, and TBC1D8 which are upregulated in cancer stem cells andassociated with the cell surface. The siRNA may target another gene inthe cell that is useful for inhibiting the growth and/or survival of thecell, for example an oncogene. For example, oncogenes that may betargeted include FOS, JUN MYB, RAS, and ABU Other exemplary oncogenesare well known in the art and several such examples are described in,for example. The Genetic Basis of Human Cancer (Vogelstein, B. andKinzler, K. W. eds. McGraw-Hill, New York. N. Y., 1998). Otherupregulated genes include Epidermal growth factor (beta-urogastrone,HOMG4/URG); Heparanase (HPA/HPR1); Jagged 1 (Alagille syndrome,AGS/AHD); Platelet/endothelial cell adhesion molecule (CD31 antigen,CD31/PECAM-1); Transforming growth factor, alpha (TFGA); and Vascularendothelial growth factor C (Flt4-L/VRP). Homologues of such genes canalso be used.

Various strategies for gene knockdown known in the art can be used toinhibit gene expression (e.g., expression of CSC-associated genes). Forexample, gene knockdown strategies may be used that make use of RNAinterference (RNAi) and/or microRNA (miRNA) pathways including smallinterfering RNA (siRNA), short hairpin RNA (shRNA), double-stranded RNA(dsRNA), miRNAs, and other small interfering nucleic acid-basedmolecules known in the art. In one embodiment, vector-based RNAimodalities (e.g., shRNA or shRNA-mir expression constructs) are used toreduce expression of a gene (e.g., a CSC-associated) in a cell. In someembodiments, therapeutic compositions of the invention comprise anisolated plasmid vector (e.g., any isolated plasmid vector known in theart or disclosed herein) that expresses a small interfering nucleic acidsuch as an shRNA. The isolated plasmid may comprise a tumor-specific,e.g., melanoma-specific, promoter operably linked to a gene encoding thesmall interfering nucleic acid, e.g., an shRNA. In some cases, theisolated plasmid vector is packaged in a virus capable of infecting theindividual. Exemplary viruses include adenovirus, retrovirus,lentivirus, adeno-associated virus, and others that are known in the artand disclosed herein.

A broad range of RNAi-based modalities could be employed to inhibitexpression of a gene in a cell, such as siRNA-based oligonucleotidesand/or altered siRNA-based oligonucleotides. Altered siRNA basedoligonucleotides are those modified to alter potency, target affinity,safety profile and/or stability, for example, to render them resistantor partially resistant to intracellular degradation. Modifications, suchas phosphorothioates, for example, can be made to oligonucleotides toincrease resistance to nuclease degradation, binding affinity and/oruptake. In addition, hydrophobization and bioconjugation enhances siRNAdelivery and targeting (De Paula et al., RNA. 13(4):431-56, 2007) andsiRNAs with ribo-difluorotoluyl nucleotides maintain gene silencingactivity (Xia et al., ASC Chem. Biol. 1(3):176-83, (2006)). siRNAs withamide-linked oligoribonucleosides have been generated that are moreresistant to S1 nuclease degradation than unmodified siRNAs (Iwase R etal. 2006 Nucleic Acids Symp Ser 50: 175-176). In addition, modificationof siRNAs at the 2′-sugar position and phosphodiester linkage confersimproved serum stability without loss of efficacy (Choung et al.,Biochem. Biophys. Res. Commun. 342(3):919-26, 2006). Other moleculesthat can be used to inhibit expression of a gene (e.g., a CSC-associatedgene) include sense and antisense nucleic acids (single or doublestranded), ribozymes, peptides, DNAzymes, peptide nucleic acids (PNAs),triple helix forming oligonucleotides, antibodies, and aptamers andmodified form(s) thereof directed to sequences in gene(s), RNAtranscripts, or proteins. Antisense and ribozyme suppression strategieshave led to the reversal of a tumor phenotype by reducing expression ofa gene product or by cleaving a mutant transcript at the site of themutation (Carter and Lemoine Br. J. Cancer. 67(5):869-76, 1993; Lange etal., Leukemia. 6(11):1786-94, 1993; Valera et al., J. Biol. Chem.269(46):28543-6, 1994; Dosaka-Akita et al., Am. J. Clin. Pathol.102(5):660-4, 1994; Feng et al., Cancer Res. 55(10):2024-8, 1995;Quattrone et al., Cancer Res. 55(1):90-5, 1995; Lewin et al., Nat Med.4(8):967-71, 1998). Ribozymes have also been proposed as a means of bothinhibiting gene expression of a mutant gene and of correcting the mutantby targeted trans-splicing (Sullenger and Cech Nature 371(6498):619-22,1994; Jones et al., Nat. Med. 2(6):643-8, 1996). Ribozyme activity maybe augmented by the use of, for example, non-specific nucleic acidbinding proteins or facilitator oligonucleotides (Herschlag et al., EmboJ. 13(12):2913-24, 1994; Jankowsky and Schwenzer Nucleic Acids Res.24(3):423-9, 1996). Multitarget ribozymes (connected or shotgun) havebeen suggested as a means of improving efficiency of ribozymes for genesuppression (Ohkawa et al., Nucleic Acids Symp Ser. (29):121-2, 1993).

Triple helix approaches have also been investigated forsequence-specific gene suppression. Triple helix formingoligonucleotides have been found in some cases to bind in asequence-specific manner (Postel et al., Proc. Natl. Acad. Sci. U.S.A.88(18):8227-31, 1991; Duval-Valentin et al., Proc. Natl. Acad. Sci.U.S.A. 89(2):504-8, 1992; Hardenbol and Van Dyke Proc. Natl. Acad. Sci.U.S.A. 93(7):2811-6, 1996; Porumb et al., Cancer Res. 56(3):515-22,1996). Similarly, peptide nucleic acids have been shown to inhibit geneexpression (Hanvey et al., Antisense Res. Dev. 1(4):307-17, 1991;Knudsen and Nielson Nucleic Acids Res. 24(3):494-500, 1996; Taylor etal., Arch. Surg. 132(11):1177-83, 1997). Minor-groove binding polyamidescan bind in a sequence-specific manner to DNA targets and hence mayrepresent useful small molecules for suppression at the DNA level(Trauger et al., Chem. Biol. 3(5):369-77, 1996). In addition,suppression has been obtained by interference at the protein level usingdominant negative mutant peptides and antibodies (Herskowitz Nature329(6136):219-22, 1987; Rimsky et al., Nature 341(6241):453-6, 1989;Wright et al., Proc. Natl. Acad. Sci. U.S.A. 86(9):3199-203, 1989). Insome cases suppression strategies have led to a reduction in RNA levelswithout a concomitant reduction in proteins, whereas in others,reductions in RNA have been mirrored by reductions in protein. Thediverse array of suppression strategies that can be employed includesthe use of DNA and/or RNA aptamers that can be selected to target aprotein of interest (e.g, a CSC-associated polypeptide).

Methods of delivering a therapeutic agent to a cancer stem cell are alsoprovided. The methods involve a step of contacting a cancer stem cellwith an isolated molecule that selectively binds to a cell surfacepolypeptide encoded by a CSC-associated gene, such as those selectedfrom the group set forth in Table 4. The CSC-associated gene may beselected from the group consisting of: ANK2, NCKAP1L, PTPRE, PTPRS,SBF1, SCN3A, SGCA, SGCB, SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8. Thecancer stem cell may be in vivo or in vitro. Isolated molecules thatbind to CSC-associated polypeptides on the surface of a cancer stem cellmay be taken up into an intracellular compartment of the cancer stemcell.

Cancer stem cells include stem cells of a colon carcinoma, a pancreaticcancer, a breast cancer, an ovarian cancer, a prostate cancer, asquamous cell carcinoma, a cervical cancer, a lung carcinoma, a smallcell lung carcinoma, a bladder carcinoma, a squamous cell carcinoma, abasal cell carcinoma, an adenocarcinoma, a sweat gland carcinoma, asebaceous gland carcinoma, a papillary carcinoma, a papillaryadenocarcinoma, a cystadenocarcinoma, a medullary carcinoma, abronchogenic carcinoma, a renal cancer, e.g., renal cell carcinoma, ahepatocellular carcinoma, a bile duct carcinoma, a choriocarcinoma, aseminoma, a embryonal carcinoma, a Wilms' tumor, or a testicular tumor.In specific embodiments, the cancer stem cells are stem cells ofmelanoma. Cancer stem cells include ABCB5⁺ cells and ABCB5⁻ cells. Stemcells of other cancers will be known to one of ordinary skill in theart.

The treatment methods of the invention involve administeringcompositions that comprise isolated molecules, or combinations ofdifferent isolated molecules, that specifically bind to CSC-associatedpolypeptides (polypeptides encoded by the CSC-associated gene) to treatcancer, e.g., melanoma, in an individual. When the CSC-associatedpolypeptide is associated with the extracellular surface of a cell,e.g., a cancer stem cell, e.g., a melanoma stem cell, the isolatedmolecule can bind the CSC-associated polypeptide and, for example, serveas an vehicle for specifically targeting therapeutic agents (therapeuticmolecules) to the cell. In some embodiments, isolated molecules thatbinds to a CSC-associated polypeptide on the surface of a cell are takenup into an intracellular compartment of the cell bind to a secretedmolecule, such as a growth factor that may assist the tumor (such asthose listed in Table 1.2). The isolated molecules that interact withunregulated proteins may be used alone as therapeutics or in combinationwith other therapeutics.

As used herein treatment of or treating cancer includes preventing thedevelopment of a cancer, reducing the symptoms of a cancer and/orinhibiting, slowing the growth of or preventing further growth of anexisting cancer. Treatment may include amelioration, cure, and/ormaintenance of a cure (i.e., prevention or delay of relapse) of adisorder, e.g., cancer. Treatment after a disorder has started aims toreduce, ameliorate or altogether eliminate the disorder, and/or itsassociated symptoms, to prevent it from becoming worse, to slow the rateof progression, or to prevent the disorder from re-occurring once it hasbeen initially eliminated (i.e., to prevent a relapse).

Cancers include for instance a colon carcinoma, a pancreatic cancer, abreast cancer, an ovarian cancer, a prostate cancer, a squamous cellcarcinoma, a cervical cancer, a lung carcinoma, a small cell lungcarcinoma, a bladder carcinoma, a squamous cell carcinoma, a basal cellcarcinoma, an adenocarcinoma, a sweat gland carcinoma, a sebaceous glandcarcinoma, a papillary carcinoma, a papillary adenocarcinoma, acystadenocarcinoma, a medullary carcinoma, a bronchogenic carcinoma, arenal cell carcinoma, a hepatocellular carcinoma, a bile duct carcinoma,a choriocarcinoma, a seminoma, an embryonal carcinoma, a Wilms' tumor,or a testicular tumor. In certain embodiments, the cancer is melanoma.

The invention, in some aspects, relates to an isolated molecule thatselectively binds to a polypeptide encoded by a CSC-associated gene setforth in Table 4 and that is conjugated to a therapeutic agent. In someinstances, the CSC-associated gene is selected from the group consistingof: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB, SLC2A11,SLC2A8, SLC4A1, STX3, and TBC1D8. Compositions comprising the foregoingisolated molecules are also disclosed.

As used herein an “isolated molecule” is a molecule such as apolypeptide, nucleic acid, polysaccharide, drug, nucleoprotein,lipoprotein, glycoprotein, steroid, and lipid that is isolated from itsnatural environment or produced synthetically. In some embodiments, theisolated molecule is a ligand of a CSC-associated polypeptide. In otherembodiments, the isolated molecule is an antibody or antigen-bindingfragment. As disclosed herein, antibody or antigen-binding fragmentsinclude monoclonal antibodies, polyclonal antibodies, human antibodies,chimeric antibodies, humanized antibodies, single-chain antibodies,F(ab′)₂, Fab, Fd, Fv, or single-chain Fv fragments. In some embodiments,an isolated molecule may have therapeutic utility alone and need not beconjugated to a therapeutic agent. For example, an isolated molecule maybind to a cell surface receptor that is a CSC-associated polypeptide andfunction as an antagonist or competitive inhibitor of the receptor(e.g., to inhibit a signaling pathway).

In some embodiments, isolated molecules are conjugated to therapeuticagents. As used herein, a “therapeutic agent” is a molecule such as apolypeptide, nucleic acid, polysaccharide, drug, nucleoprotein,lipoprotein, glycoprotein, steroid, and lipid that is capable ofaltering the state of a cell (e.g., killing a cell, inhibiting growth ofa cell) for therapeutic purposes. A therapeutic agent may be, forinstance, a toxin, a small-interfering nucleic acid, or achemotherapeutic agent. Alternatively the therapeutic may beadministered in conjunction with the molecule. In conjunction refers todelivery to the same subject. The actual administration may be at thesame or a different time or in the same or a different delivery vehicle.

Toxins include for example, radioisotopes such as ²²⁵Ac, ²¹¹At, ²¹²Bi,²¹³Bi, ¹⁸⁶Rh, ¹⁸⁸Rh, ¹⁷⁷Lu, ⁹⁰Y, ¹³¹I, ⁶⁷Cu, ¹²⁵I, ¹²³I, ⁷⁷Br, ¹⁵³Sm,¹⁶⁶Bo, ⁶⁴Cu, ²¹²Pb, ²²⁴Ra and ²²³Ra, and others known in the art.Suitable chemical toxins include members of the enediyne family ofmolecules, such as calicheamicin and esperamicin as well as poisonouslectins, plant toxins such as ricin, abrin, modeccin, botulina anddiphtheria toxins. Of course, combinations of the various toxins couldalso be coupled to one isolated molecule, e.g., an antibody, therebyaccommodating variable cytotoxicity. The coupling of one or more toxinmolecules to the isolated molecule, e.g., antibody, is envisioned toinclude many chemical mechanisms, for instance covalent binding,affinity binding, intercalation, coordinate binding, and complexation.

Chemotherapeutic agents include the following compounds or classes ofcompounds: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine;Adozelesin; Adriamycin; Aldesleukin; Altretamine; Ambomycin; AmetantroneAcetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin;Asparaginase; Asperlin; Azacitidine; Azetepa; Azotomycin; Batimastat;Buniodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate;Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan;Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin;Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol;Chlorombucil; Cirolemycin; Cisplatin; Cladribine; Crisnatol Mesylate;Cyclophosphamide; Cytarabine; Dacarbazine; DACA(N-[2-(Dimethyl-amino)ethyl]acridine-4-carboxamide); Dactinomycin;Daunorubicin Hydrochloride; Daunomycin; Decitabine; Dexormaplatin;Dezaguanine; Dezaguanine Ifesylate; Diaziquone; Docetaxel; Doxorubicin;Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate;Dromostanolone Propionate; Duazomycin; Edatrexate; EflornithineHydrochloride; Elsamitrucin; Enloplatin; Enpromate; Epipropidine;Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride;Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized OilI 131; Etoposide; Etoposide Phosphate; Etoprine; FadrozoleHydrochloride; Fazarabine; Fenretinide; Floxuridine; FludarabinePhosphate; Fluorouracil; 5-FdUMP; Flurocitabine; Fosquidone; FostriecinSodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198;Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; Ilmofosine;Interferon Alfa-2a; Interferon Alfa-2b; Interferon Alfa-n1; InterferonAlfa-n3; Interferon Beta-1a; Interferon Gamma-1b; Iproplatin; IrinotecanHydrochloride; Lanreotide Acetate; Letrozole; Leuprolide Acetate;Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; LosoxantroneHydrochloride; Masoprocol; Maytansine; Mechlorethamine Hydrochloride;Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril;Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin;Mitomycin, Mitosper; Mitotane; Mitoxantrone Hydrochloride; MycophenolicAcid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; PaclitaxelPegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate;Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride;Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine;Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride;Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin; SpirogermaniumHydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin;Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid;Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin;Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine;Thiotepa; Thymitaq; Tiazofurin; Tirapazamine; Tomudex; TOP-53; TopotecanHydrochloride; Toremifene Citrate; Trestolone Acetate; TriciribinePhosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;Verteporfin; Vinblastine; Vinblastine Sulfate; Vincristine; VincristineSulfate, Vindesine; Vindesine Sulfate; Vinepidine Sulfate; VinglycinateSulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; VinrosidineSulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin;Zorubicin Hydrochloride; 2-Chlorodeoxyadenosine; 2′-Deoxyformycin;9-aminocamptothecin; raltitrexed; N-propargyl-5, 8-dideazafolic acid,2-chloro-2′-arabino-fluoro-2′-deoxyadenosine;2-chloro-2′-deoxyadenosine; anisomycin; trichostatin A; hPRL-G129R;CEP-751; linomide; Piritrexim Isethionate; Sitogluside; TamsulosinHydrochloride and Pentomone.

The invention, in some aspects, provides kits comprising one or morecontainers housing one or more of the compositions of the invention. Thekit may be designed to facilitate use of the methods described herein byresearchers and can take many forms. Each of the compositions of thekit, where applicable, may be provided in liquid form (e.g., insolution), or in solid form, (e.g., a dry powder). In certain cases,some of the compositions may be constitutable or otherwise processable(e.g., to an active form), for example, by the addition of a suitablesolvent or other species (for example, water or a cell culture medium),which may or may not be provided with the kit. The kits may also includereference samples. As used herein, “instructions” can define a componentof instruction and/or promotion, and typically involve writteninstructions on or associated with packaging of the invention.Instructions also can include any oral or electronic instructionsprovided in any manner such that a user will clearly recognize that theinstructions are to be associated with the kit, for example, audiovisual(e.g., videotape, DVD, etc.), Internet, and/or web-based communications,etc. The written instructions may be in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which instructions can alsoreflects approval by the agency of manufacture, use or sale for animaladministration.

The kit may contain any one or more of the components described hereinin one or more containers. As an example, in one embodiment, the kit mayinclude instructions for mixing one or more components of the kit and/orisolating and mixing a sample and applying to a individual. The kit mayinclude a container housing agents described herein. The agents may bein the form of a liquid, gel or solid (powder). The agents may beprepared sterilely, packaged in syringe and shipped refrigerated.Alternatively it may be housed in a vial or other container for storage.A second container may have other agents prepared sterilely.Alternatively the kit may include the active agents premixed and shippedin a syringe, vial, tube, or other container. The kit may have one ormore or all of the components required to administer the agents to ananimal, such as a syringe, topical application devices, or iv needletubing and bag, particularly in the case of the kits for treatingindividuals with cancer.

The compositions and therapeutic agents may be administeredsimultaneously or sequentially. When the other therapeutic agents areadministered simultaneously they can be administered in the same orseparate formulations, but are administered at the same time. The othertherapeutic agents are administered sequentially with one another andwith the modulators, when the administration of the other therapeuticagents and the modulators is temporally separated. The separation intime between the administration of these compounds may be a matter ofminutes or it may be longer.

The compositions of the present invention preferably contain apharmaceutically acceptable carrier or excipient suitable for renderingthe compound or mixture administrable orally as a tablet, capsule orpill, or parenterally, intravenously, intradermally, intramuscularly orsubcutaneously, or transdermally. The active ingredients may be admixedor compounded with any conventional, pharmaceutically acceptable carrieror excipient. The compositions may be sterile.

As used herein, the term “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the compositions of this invention,its use in the therapeutic formulation is contemplated. Supplementaryactive ingredients can also be incorporated into the pharmaceuticalformulations. A composition is said to be a “pharmaceutically acceptablecarrier” if its administration can be tolerated by a recipient patient.Sterile phosphate-buffered saline is one example of a pharmaceuticallyacceptable carrier. Other suitable carriers are well-known in the art.

It will be understood by those skilled in the art that any mode ofadministration, vehicle or carrier conventionally employed and which isinert with respect to the active agent may be utilized for preparing andadministering the pharmaceutical compositions of the present invention.Illustrative of such methods, vehicles and carriers are those described,for example, in Remington's Pharmaceutical Sciences, 18th ed. (1990),the disclosure of which is incorporated herein by reference. Thoseskilled in the art, having been exposed to the principles of theinvention, will experience no difficulty in determining suitable andappropriate vehicles, excipients and carriers or in compounding theactive ingredients therewith to form the pharmaceutical compositions ofthe invention.

An effective amount, also referred to as a therapeutically effectiveamount is an amount sufficient to ameliorate at least one adverse effectassociated with expression, or reduced expression, of a CSC-associatedgene in a cell or in an individual in need of such inhibition orsupplementation. The therapeutically effective amount to be included inpharmaceutical compositions depends, in each case, upon several factors,e.g., the type, size and condition of the patient to be treated, theintended mode of administration, the capacity of the patient toincorporate the intended dosage form, etc. Generally, an amount ofactive agent is included in each dosage form to provide from about 0.1to about 250 mg/kg, and preferably from about 0.1 to about 100 mg/kg.One of ordinary skill in the art would be able to determine empiricallyan appropriate therapeutically effective amount.

Combined with the teachings provided herein, by choosing among thevarious active compounds and weighing factors such as potency, relativebioavailability, patient body weight, severity of adverse side-effectsand preferred mode of administration, an effective prophylactic ortherapeutic treatment regimen can be planned which does not causesubstantial toxicity and yet is entirely effective to treat theparticular individual. The effective amount for any particularapplication can vary depending on such factors as the disease orcondition being treated, the particular therapeutic agent beingadministered, the size of the individual, or the severity of the diseaseor condition. One of ordinary skill in the art can empirically determinethe effective amount of a particular nucleic acid and/or othertherapeutic agent without necessitating undue experimentation.

The pharmaceutical compositions can be administered by any suitableroute for administering medications. A variety of administration routesare available. The particular mode selected will depend, of course, uponthe particular agent or agents selected, the particular condition beingtreated, and the dosage required for therapeutic efficacy. The methodsof this invention, generally speaking, may be practiced using any modeof administration that is medically acceptable, meaning any mode thatproduces effective levels of an immune response without causingclinically unacceptable adverse effects. Preferred modes ofadministration are discussed herein. For use in therapy, an effectiveamount of the nucleic acid and/or other therapeutic agent can beadministered to an individual by any mode that delivers the agent to thedesired surface, e.g., mucosal, systemic.

Administering the pharmaceutical composition of the present inventionmay be accomplished by any means known to the skilled artisan. Routes ofadministration include but are not limited to oral, parenteral,intravenous, intramuscular, intraperitoneal, intranasal, sublingual,intratracheal, inhalation, subcutaneous, ocular, vaginal, and rectal.Systemic routes include oral and parenteral. Several types of devicesare regularly used for administration by inhalation. These types ofdevices include metered dose inhalers (MDI), breath-actuated MDI, drypowder inhaler (DPI), spacer/holding chambers in combination with MDI,and nebulizers.

In some cases, compounds of the invention are prepared in a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvesicles (LUVs), which range in size from 0.2-4.0 μm can encapsulatelarge macromolecules. RNA, DNA and intact virions can be encapsulatedwithin the aqueous interior and be delivered to cells in a biologicallyactive form. Fraley et al. (1981) Trends Biochem Sci 6:77.

Liposomes may be targeted to a particular tissue by coupling theliposome to a specific binding molecule such as one that binds to aCSC-associated polypeptide. Binding molecules which may be useful fortargeting a liposome to, for example, a cancer stem cell include, butare not limited to intact or fragments of molecules, e.g., antibodies orantigen binding fragments, which interact with CSC-associatedpolypeptides on the surface of cancer stem cells. Such binding moleculesmay easily be identified by binding assays well known to those of skillin the art.

Lipid formulations for transfection are commercially available fromQIAGEN, for example, as EFFECTENE™ (a non-liposomal lipid with a specialDNA condensing enhancer) and SUPERFECT™ (a novel acting dendrimerictechnology).

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTIN™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2, 3 dioleyloxy)-propyl]-N, N, N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis G(1985) Trends Biotechnol 3:235-241.

Certain cationic lipids, including in particular N-[1-(2, 3dioleoyloxy)-propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP),appear to be especially advantageous when combined with the modifiedoligonucleotide analogs of the invention.

In one embodiment, the vehicle is a biocompatible microparticle orimplant that is suitable for implantation or administration to themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US/03307 (Publication No. WO95/24929, entitled“Polymeric Gene Delivery System”. PCT/US/0307 describes a biocompatible,preferably biodegradable polymeric matrix for containing an exogenousgene under the control of an appropriate promoter. The polymeric matrixcan be used to achieve sustained release of the therapeutic agent in theindividual.

The polymeric matrix preferably is in the form of a microparticle suchas a microsphere (e.g., wherein a therapeutic agent is dispersedthroughout a solid polymeric matrix) or a microcapsule (e.g., wherein atherapeutic agent is stored in the core of a polymeric shell). Otherforms of the polymeric matrix for containing the therapeutic agentinclude films, coatings, gels, implants, and stents. The size andcomposition of the polymeric matrix device is selected to result infavorable release kinetics in the tissue into which the matrix isintroduced. The size of the polymeric matrix further is selectedaccording to the method of delivery which is to be used, typicallyinjection into a tissue or administration of a suspension by aerosolinto the nasal and/or pulmonary areas. Preferably when an aerosol routeis used the polymeric matrix and the therapeutic agent are encompassedin a surfactant vehicle. The polymeric matrix composition can beselected to have both favorable degradation rates and also to be formedof a material which is bioadhesive, to further increase theeffectiveness of transfer when the matrix is administered to a nasaland/or pulmonary surface that has sustained an injury. The matrixcomposition also can be selected not to degrade, but rather, to releaseby diffusion over an extended period of time. Biocompatible microspheresthat are suitable for delivery, such as oral or mucosal delivery, aredisclosed in Chickering et al. (1996) Biotech Bioeng 52:96-101 andMathiowitz E et al. (1997) Nature 386:410-414 and PCT Pat. ApplicationWO97/03702.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the therapeutic agentsto an individual. Biodegradablematrices are preferred. Such polymers may be natural or syntheticpolymers. The polymer is selected based on the period of time over whichrelease is desired, generally in the order of a few hours to a year orlonger. Typically, release over a period ranging from between a fewhours and three to twelve months is most desirable, particularly for thenucleic acid agents. The polymer optionally is in the form of a hydrogelthat can absorb up to about 90% of its weight in water and further,optionally is cross-linked with multi-valent ions or other polymers.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, (1993) 26:581-587, the teachings of which areincorporated herein. These include polyhyaluronic acids, casein,gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan,poly(methyl methacrylates), poly(ethyl methacrylates),poly(butylmethacrylate), poly(isobutyl methacrylate),poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(laurylmethacrylate), poly(phenyl methacrylate), poly(methyl acrylate),poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecylacrylate).

If the therapeutic agent is a nucleic acid, the use of compaction agentsmay also be desirable. Compaction agents also can be used alone, or incombination with, a biological or chemical/physical vector. A“compaction agent”, as used herein, refers to an agent, such as ahistone, that neutralizes the negative charges on the nucleic acid andthereby permits compaction of the nucleic acid into a fine granule.Compaction of the nucleic acid facilitates the uptake of the nucleicacid by the target cell. The compaction agents can be used alone, i.e.,to deliver a nucleic acid in a form that is more efficiently taken up bythe cell or, more preferably, in combination with one or more of theabove-described vectors.

Other exemplary compositions that can be used to facilitate uptake of anucleic acid include calcium phosphate and other chemical mediators ofintracellular transport, microinjection compositions, electroporationand homologous recombination compositions (e.g., for integrating anucleic acid into a preselected location within the target cellchromosome).

The compounds may be administered alone (e.g., in saline or buffer) orusing any delivery vehicle known in the art. For instance the followingdelivery vehicles have been described: cochleates; Emulsomes; ISCOMs;liposomes; live bacterial vectors (e.g., Salmonella, Escherichia coli,Bacillus Calmette-Guérin, Shigella, Lactobacillus); live viral vectors(e.g., Vaccinia, adenovirus, Herpes Simplex); microspheres; nucleic acidvaccines; polymers (e.g. carboxymethylcellulose, chitosan); polymerrings; proteosomes; sodium fluoride; transgenic plants; virosomes; and,virus-like particles.

The formulations of the invention are administered in pharmaceuticallyacceptable solutions, which may routinely contain pharmaceuticallyacceptable concentrations of salt, buffering agents, preservatives,compatible carriers, adjuvants, and optionally other therapeuticingredients.

The term pharmaceutically-acceptable carrier means one or morecompatible solid or liquid filler, diluents or encapsulating substanceswhich are suitable for administration to a human or other vertebrateanimal. The term carrier denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being commingled with the compounds ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficiency.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by an individual to be treated. Pharmaceutical preparationsfor oral use can be obtained as solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. Optionally the oralformulations may also be formulated in saline or buffers forneutralizing internal acid conditions or may be administered without anycarriers.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. Microspheres formulatedfor oral administration may also be used. Such microspheres have beenwell defined in the art. All formulations for oral administration shouldbe in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention may be conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of e.g. gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with an added preservative. The compositions may take suchforms as suspensions, solutions or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal or vaginal compositionssuch as suppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long-acting formulationsmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, forexample, aqueous or saline solutions for inhalation, microencapsulated,encochleated, coated onto microscopic gold particles, contained inliposomes, nebulized, aerosols, pellets for implantation into the skin,or dried onto a sharp object to be scratched into the skin. Thepharmaceutical compositions also include granules, powders, tablets,coated tablets, (micro)capsules, suppositories, syrups, emulsions,suspensions, creams, drops or preparations with protracted release ofactive compounds, in whose preparation excipients and additives and/orauxiliaries such as disintegrants, binders, coating agents, swellingagents, lubricants, flavorings, sweeteners or solubilizers arecustomarily used as described above. The pharmaceutical compositions aresuitable for use in a variety of drug delivery systems. For a briefreview of methods for drug delivery, see Langer R (1990) Science249:1527-1533, which is incorporated herein by reference.

The compounds may be administered per se (neat) or in the form of apharmaceutically acceptable salt. When used in medicine the salts shouldbe pharmaceutically acceptable, but non-pharmaceutically acceptablesalts may conveniently be used to prepare pharmaceutically acceptablesalts thereof. Such salts include, but are not limited to, thoseprepared from the following acids: hydrochloric, hydrobromic, sulphuric,nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulphonic,tartaric, citric, methane sulphonic, formic, malonic, succinic,naphthalene-2-sulphonic, and benzene sulphonic. Also, such salts can beprepared as alkaline metal or alkaline earth salts, such as sodium,potassium or calcium salts of the carboxylic acid group.

Suitable buffering agents include: acetic acid and a salt (1-2% w/v);citric acid and a salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v);and phosphoric acid and a salt (0.8-2% w/v). Suitable preservativesinclude benzalkonium chloride (0.003-0.03% w/v); chlorobutanol (0.3-0.9%w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-0.02% w/v).

The compositions may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the compounds into associationwith a carrier which constitutes one or more accessory ingredients. Ingeneral, the compositions are prepared by uniformly and intimatelybringing the compounds into association with a liquid carrier, a finelydivided solid carrier, or both, and then, if necessary, shaping theproduct. Liquid dose units are vials or ampoules. Solid dose units aretablets, capsules and suppositories.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compounds, increasing convenience to theindividual and the physician. Many types of release delivery systems areavailable and known to those of ordinary skill in the art. They includepolymer base systems such as poly(lactide-glycolide), copolyoxalates,polycaprolactones, polyesteramides, polyorthoesters, polyhydroxybutyricacid, and polyanhydrides. Microcapsules of the foregoing polymerscontaining drugs are described in, for example, U.S. Pat. No. 5,075,109.Delivery systems also include non-polymer systems that are: lipidsincluding sterols such as cholesterol, cholesterol esters and fattyacids or neutral fats such as mono-, di-, and tri-glycerides; hydrogelrelease systems; silastic systems; peptide-based systems; wax coatings;compressed tablets using conventional binders and excipients; partiallyfused implants; and the like. Specific examples include, but are notlimited to: (a) erosional systems in which an agent of the invention iscontained in a form within a matrix such as those described in U.S. Pat.Nos. 4,452,775, 4,675,189, and 5,736,152, and (b) diffusional systems inwhich an active component permeates at a controlled rate from a polymersuch as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.In addition, pump-based hardware delivery systems can be used, some ofwhich are adapted for implantation.

EXAMPLES

Among the numerous CSC-associated genes are genes involved invasculogenesis and angiogenesis. For example, global gene expressionanalyses validated by mRNA and protein determinations revealedpreferential display of genes for vascular endothelial growth factorreceptor-1 (VEGFR-1) and related members of signaling cascades involvedin vasculogenesis and angiogenesis in ABCB5⁺ MMIC. In vitro, vascularendothelial growth factor (VEGF) induced expression of theendothelial-associated marker CD144 (VE-cadherin) in VEGFR-1-expressingABCB5⁺ MMIC but not VEGFR-1-negative ABCB5⁻ melanoma bulk populations,indicating a unique capacity of CSC for VEGF/VEGFR-1 signaling-dependentvasculogenic differentiation. In vivo, tumors initiated frompatient-derived melanoma cells or established melanoma cultures byxenotransplantation into the murine subcutis or by intradermal injectioninto human skin in chimeric murine recipients formed perfused ABCB5mRNA- and protein-expressing vessel-like channels also detected inclinical melanoma specimens that co-expressed CD144 and the vasculogenicmimicry marker TIE-1⁴. Tumour initiation in human skin by fluorescenttransgene-expressing human melanoma cells confirmed CD144⁺ channels tobe of melanoma origin. Moreover, MMIC depletion in tumour grafts tohuman skin significantly reduced channel formation and resulted inattenuated tumour growth. Our results identify melanoma vasculogenesisdriven by ABCB5⁺ MMIC as a novel mechanism by which CSC may promotetumor growth. Furthermore, they suggest that MMIC-dependentvasculogenesis represents a novel CSC target for VEGF/VEGFR-1-directedinhibitors of angiogenesis.

Example 1 Materials and Methods

Melanoma Cells and Culture Methods.

The established human cutaneous melanoma cell lines G3361, A375,MALME-3M, SK-MEL-2, SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14 andMDA-MB-435 were cultured as described^(3,5,23). Clinical cutaneousmelanoma cells were derived from surgical specimen according to humansubjects research protocols approved by the IRBs of the University ofWürzburg Medical School or the Wistar Institute, Philadelphia, Pa. asdescribed previously³. The established human uveal melanoma cell linesMUM-2B and MUM-2C were a gift of Dr. Mary J. Hendrix, NorthwesternUniversity, and were cultured as described¹⁶.

Cell Isolation.

ABCB5⁺-purified (ABCB5⁺) cells were isolated by positive selection andABCB5⁺-depleted (ABCB5⁻) cell populations were generated by removingABCB5⁺ cells using anti-ABCB5 mAb (clone 3C2-1D12²³) labeling andmagnetic bead cell sorting as described³. Briefly, human G3361, A375,MUM-2B or MUM-2C melanoma cells or single cell suspensions derived fromclinical melanoma samples were labeled with anti-ABCB5 mAb for 30 min at4° C., washed twice for excess antibody removal, followed by incubationwith secondary anti-mouse IgG mAb-coated magnetic microbeads for 30 minat 4° C. Subsequently, cells were washed twice for excess magneticmicrobead removal and then sorted into ABCB5⁺ and ABCB5⁻ cell fractionsby dual-passage cell separation in MidiMACS or MiniMACS separationcolumns (depending on cell number) according to the manufacturer'srecommendations (Miltenyi Biotec, Auburn, Calif.). Assessment of purityof ABCB5⁺ and ABCB5⁻ (ABCB5⁺ cell-depleted) melanoma cell isolates anddetermination of cell viability following magnetic cell sorting wereperformed and yielded similar results as described previously³.

Global Gene Expression Microarray Analyses.

Microarray analyses were performed on purified ABCB5⁺ (n=5) and ABCB5⁻(n=5) cell subsets derived from the established human melanoma celllines G3361 and A375 and from three distinct clinical melanoma specimenpreviously characterized in our laboratory with regards to ABCB5expression and MMIC phenotype in human melanoma xenotransplantationassays³. Total RNA was extracted, processed and hybridized as describedpreviously¹⁰ onto Affymetrix human HG-U133Plus2 GeneChip microarrays(Affymetrix, Santa Clara, Calif.). Statistical analysis of microarrayresults was performed as described previously¹⁰. The expression data setin its entirety will be made available through GEO (gene expressionomnibus). Functional gene networks were generated using IngenuityPathways Analysis (Ingenuity® Systems, ingenuity.com), by mapping eachgene identifier to its corresponding gene object in the IngenuityPathways Knowledge Base. These focus genes were overlaid onto a globalmolecular network developed from information contained in the IngenuityPathways Knowledge Base. Focus gene networks were then algorithmicallygenerated based on their connectivity and subsequently analyzed toidentify the biological functions that were most significant to thegenes in the network.

RNA Extraction and Reverse Transcriptase-PCR.

Total RNA was isolated from ABCB5⁺ and ABCB5⁻ human melanoma cells usingRNeasy columns (QIAGEN, Valencia, Calif.). Standard cDNA synthesisreactions were performed using 5 μg RNA and the SuperScript First-StrandSynthesis System for reverse transcriptase-PCR (Invitrogen, Carlsbad,Calif.) as per the manufacturer's instructions. For PCR analysis, 5 μlof diluted first strand product (˜100 ng of cDNA) was added to 45 μl ofPCR reaction mixture containing 5 units of Superscript II (Invitrogen)according to the manufacturer's protocol. The following PCR program wasperformed: denaturation at 95° C. for 5 min, then cycled 35 times at 94°C. for 1 min, 53° C. for 30 s, and 72° C. for 30 s, and subsequentlyextended at 72° C. for 10 min. The primer sequences were as follows:PTK2 forward primer, 5′-TGCCTTTTACTTTCGTGTGG-3′(SEQ ID NO:1); PTK2reverse primer 5′-CCAAATTCCTGTTTTGCTTCA-3′(SEQ ID NO:2); MET forwardprimer 5′-CCCCACCTTATCCTGACGTA-3′(SEQ ID NO:3); MET reverse primer5′-CGTGTGTCCACCTCATCATC-3′(SEQ ID NO:4); NRP2 forward primer5′-GAGGCAGGGGAAAATAGAGG-3′(SEQ ID NO:5); NRP2 reverse primer5′-TCTCCCGAAAGGTTGAAATG-3′(SEQ ID NO:6); ETS1 forward primer5′-CGCTTACTCTGTTGGGGTCT-3′(SEQ ID NO:7); ETS1 reverse primer5′-TCTCCAGCAAAATGATGTGC-3′(SEQ ID NO:8); FLT1 forward primer5′-TGGCAACTGCTTTTATGTTCTG-3′(SEQ ID NO:9); FLT1 reverse primer5′-TCCATAGGGTGATGGTCAAA-3′(SEQ ID NO: 10). The reaction products wereresolved on a 1% LE agarose gel (Ambion, Austin, Tex.) and photographed.β-Actin primers were used as controls to ensure RNA integrity.

RNA Extraction and Real Time Quantitative PCR.

Total RNA was isolated from unsegregated or sorted human melanoma cellsusing the RT² qPCR Grade RNA isolation kit (SABiosciences, Frederick,Md.). Standard cDNA synthesis reactions were performed using 1 μg RNAand the RT² First Strand Kit for reverse transcriptase-PCR(SABiosciences) as per the manufacturer's instructions. The reversetranscriptase product (1 μl) was amplified by primer pairs specific forABCB5⁵. β-actin was used as a normalizing control. The primers for ABCB5(Genebank accession no. AY234788) detection were5′-GCTGAGGAATCCACCCAATCT-3′ (forward) (SEQ ID NO:11) and5′-AGCCTGAATGGCCTTTTGTG-3′ (reverse) (SEQ ID NO:12). The primers forβ-actin detection were 5′-CCTGGCACCCAGCACAAT-3′ (SEQ ID NO:13) (forward)and 5′-AGTACTCCGTGTGGATCGGC-3′ (reverse) (SEQ ID NO: 14). Samples wereassayed using Sybergreen chemistry and kinetic PCR (ABI 7300 SequenceDetector; Applied Biosystems, Foster City, Calif.). Samples wereamplified using the Sybergreen PCR reagent kit (Applied Biosystems)according to the manufacturer's protocol. Sense and antisense primerswere used at a final concentration of 10 nM. The cDNA samples wereamplified under following conditions: 50° C. for 2 min and 95° C. for 10min, followed by 40 cycles of amplification at 94° C. for 15 s and 60°C. for 1 min. All samples were run in triplicate. The relative amountsof transcripts were analyzed using the 2(−Delta Delta C(T)) method asdescribed previously^(3,5,10). Statistical differences between mRNAexpression levels were determined using the nonparametric Mann-Whitneytest. A two-sided P value of P<0.05 was considered significant.

Western Analysis.

Total cell lysates were harvested from logarithmically growing culturesof the human melanoma cell lines MALME-3M, SK-MEL-2, SK-MEL-5,SK-MEL-28, UACC-62, UACC-257, M14, and MDA-MB-435 and analyzed by 8%SDS-PAGE and Western assay to detect relative levels of ABCB5 (mAb3C2-1D12²³) and alpha-tubulin (mAb clone DM1A, Sigma-Aldrich, St. Louis,Mo.), using LI-COR Odyssey IR imaging system densitometry.

Flow Cytometry.

G3361, A375, MUM-2B, or MUM-2C melanoma cells were analyzed for surfaceABCB5 expression by incubation with anti-ABCB5 mAb or isotype controlmAb for 30 min at 4° C., followed by counterstaining withFITC-conjugated goat anti-mouse Ig or with APC-conjugated donkeyanti-mouse IgG secondary Abs and single color flow cytometry at the Fl1(FITC) or F14 (APC) emission spectrum on a Becton Dickinson FACScan asdescribed^(3,5,23). Washing was performed twice between each step.Analysis of coexpression of ABCB5 with the VEGFR-1 surface marker inG3361 melanoma cells was performed by dual-color flow cytometry asdescribed³. Briefly, melanoma cells were incubated for 30 min at 4° C.with anti-ABCB5 mAb or isotype control mAb, followed by counterstainingwith APC-conjugated donkey anti-mouse IgG secondary Ab as above.Subsequently, cells were fixed at 4° C. and then incubated for 30 min at4° C. with PE-conjugated anti-VEGFR-1 mAb (R&D Systems, Minneapolis,Minn.) or PE-conjugated isotype control mAb (BD PharMingen, San Diego,Calif.). Dual color flow cytometry was subsequently performed withacquisition of fluorescence emission at the F14 (APC) and F12 (PE)spectra on a Becton Dickinson FACScan. Washing was performed twicebetween each step. Statistical differences between expression levels ofmarkers were determined using the nonparametric Mann-Whitney test. Atwo-sided P value of P<0.05 was considered significant.

In Vitro Vasculogenic Differentiation and Tube Formation Assays.

VEGF-dependent induction of CD144 expression and formation ofcapillary-like tube structures by human G3361 melanoma cells was assayedon growth factor reduced Matrigel, a basement membrane matrixpreparation (BD Biosciences, San Jose, Calif.). Growth factor reducedMatrigel was added to eight-chamber polystyrene vessel tissueculture-treated glass slides and allowed to gelatinize for 20 min at 37°C. Purified ABCB5⁺ or ABCB5⁻ or unsegregated human melanoma cells wereseeded into culture slide wells at densities of 5×10⁴ cells/cm² inmedium 199 containing 5% FCS¹¹ in the presence or absence of VEGF (100ng/ml). After 48-hour incubation, cells were fixed with 4%paraformaldehyde/PBS for 20 min at room temperature, and after extensivewashing with PBS the cells were blocked in 5% donkey serum/0.01% Tween20/PBS for 1 hr at room temperature. Cells were then incubated withrabbit anti-CD144 polyclonal Ab (diluted 1:100; Bethyl Laboratories,Montgomery, Tex.) overnight at 4° C. After extensive washing with 0.01%Tween 20/PBS, the cells were incubated with goat anti-rabbit Texasred-conjugated secondary Ab (diluted 1:250; Jackson ImmunoResearchLaboratories, West Grove, Pa.) for 1 hr at room temperature. Followingwashing with 1×PBS/0.01% Tween 20, cells were then mounted inVectashield (Vecta Laboratories, Burlingame, Calif.) supplemented with100 ng/ml DAPI to visualize nuclei. Cells were analyzed by fluorescentmicroscopy using a Mercury-100 Watts fluorescent light source(Microvideo Instruments, Avon, Mass.) attached to a Nikon Eclipse TE 300microscope (Nikon Instruments, Melville, N.Y.) with the use of separatefilters for each fluorochrome. The images were obtained using a Spotdigital camera (Diagnostic Instruments Inc., Sterling Heights, Mich.),and the Spot 3.3.2. software package was imported into Adobe Photoshop(Adobe Systems, Mountain View, Calif.). For tube formation assays,unsegregated human melanoma cells were seeded into culture slide wellsat densities of 5×10⁴ cells/cm² in medium 199 containing 5% FCS¹¹.Immediately, cells were pretreated with medium alone, rabbit anti-VEGFR1Ab (10 μg/ml; Santa Cruz Biotechnology, Santa Cruz, Calif.) or rabbitisotype control Ab (10 μg/ml; BD Biosciences) at 37° C. for 2 hrs priorto stimulation with VEGF (100 ng/ml). Tube formation was detected byphase contrast microscope (Nikon Eclipse TE 300 microscope) after 24 hrsof incubation. For quantitative analysis of tube formation and lengthand for determination of CD144 expression at 48 hrs, n=3 three randomlyselected microscopy fields were photographed per experimental condition.Images were acquired as described above and tube formation was analyzedusing Image J software available from the National Institutes of Healthweb site as described previously²⁴. For quantification of CD144expression, positive cells were counted using Neurolucida 8.10 software(MBF Bioscience, Williston, Vt.). Differences among groups were analyzedby one-way ANOVA followed by Bonferroni post hoc tests. Differences withP values<0.05 were considered statistically significant.

In Vitro Adipogenic, Osteogenic and Myogenic Differentiation Assays.

For adipogenic, osteogenic and myogenic differentiation assays purifiedABCB5⁺ and ABCB5⁻ G3361 melanoma cells were seeded in triplicate at3×10³ cells/well in 96-well culture plates. Adipogenic and osteogenicdifferentiation was assessed using commercially availabledifferentiation kits and Oil Red O and Alizarin Red staining,respectively, according to the manufacturer's instructions (ChemiconInternational, Temecula, Calif.). Myogenic differentiation assays wereperformed as described previously¹⁰. Briefly, melanoma subpopulationswere incubated in growth medium consisting of DMEM with 4% glucose, 20%fetal bovine serum (vol/vol), 1% (vol/vol) penicillin-streptomycin(10,000 UI/ml-10,000 μg/ml, Invitrogen) for 10 days. The medium wasexchanged every 2 days. At day 14, cells were fixed in ice cold methanolfor 3 min on ice and incubated with 1:50 diluted anti-myogenin mousemonoclonal Ab (Dako, Carpinteria, Calif.) overnight at 4° C. Plates werethen washed, incubated with goat anti-mouse FITC-conjugated secondary Ab(Jackson ImmunoResearch Laboratories, diluted 1:100) and then mountedwith Vectashield mounting medium (Vector) supplemented with 100 ng/mlDAPI to visualize nuclei. Slides were visualized with the 20× and 40×objective on a Nikon Eclipse TE2000-S microscope, photographed using theSpot 7.4 slider camera and images processed using Spot software version4.0.9. (Diagnostic Instruments, Sterling Heights, Mich.). Forquantification of differentiation marker expression, positive cells weremanually counted and statistical differences between expression levelsof markers were determined using the nonparametric Mann-Whitney test. Atwo-sided P value of P<0.05 was considered significant.

Immunohistochemistry and Immunofluorescence Double Labeling.

The following primary Abs were used: rat anti-Laminin B2, (Abcam,Cambridge, Mass.), rabbit anti-CD144, (Cell Signaling, Beverly, Mass.),goat anti-Tie-1, (Neuromics, Edina, Minn.), and mouseanti-ABCB5^(3,5,23). Isotype matched irrelevant Abs served as negativecontrol. The secondary Abs were horse anti-mouse IgG-HRP, horseanti-goat IgG-HRP, goat anti-rabbit IgG-HRP (VECTOR Laboratories,Burlingame, Calif.) and goat anti-rat IgG-HRP (Biolegend, San Diego,Calif.), and donkey anti-mouse IgG-AF488, donkey anti-rabbit IgG-AF594,and donkey anti-goat IgG-AF594 (Invitrogen, Carlsbad, Calif.).Immunohistochemistry was performed using the 2-step horseradishperoxidase method. Briefly, frozen tissue sections were fixed with −20°C. acetone for 5 min, then incubated with primary Ab at 4° C. overnight.After washing out unbound primary Ab with phosphate-buffered saline(PBS), the tissue sections were incubated with secondary Ab at roomtemperature for 30 min, then washed with PBS 3×5 min. Immunoreactivitywas detected using NovaRed peroxidase substrate (VECTOR Laboratories,Burlingame, Calif.). For immunofluorescence double labeling, the frozentissue sections were fixed with −20° C. acetone for 5 min, thenincubated with the mix of 2 primary Abs (for example ABCB5 Ab+CD144 Ab)at 40° C. overnight. After washing with PBS, the tissue sections wereincubated with the mix of 2 secondary Abs (for example donkey anti-mouseIgG-AF488+ donkey anti-rabbit IgG-AF594) at room temperature for 1 hour,then washed with PBS 3×5 min, and the sections were then mounted withProLong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, Calif.).The sections were viewed under a Olympus BX51 System fluorescencemicroscope (Olympus Corporation, Tokyo, Japan). For HLA-2Aimmunohistochemistry of SK-MEL-5 melanoma xenografts to chimericmouse/human skin, frozen sections were incubated with 5 μg/ml mouseanti-human HLA-2A Ab (BD Pharmingen, San Jose, Calif.) at 4° C.overnight. After washing out unbound primary Ab with phosphate bufferedsaline (PBS), sections were incubated with 1:200 peroxidase-conjugatedhorse anti-mouse IgG Ab (Vector Laboratories, Burlingame, Calif.) atroom temperature for 30 min. Unbound secondary Ab was washed out withPBS. Color was developed using the NovRed peroxidase substrate kit(Vector Laboratories) and sections were counterstained with hematoxylinGill's No.1 (Fisher Scientific, Pittsburgh, Pa.).

In Situ Hybridization.

RNA probes were prepared as follows: PCR-derived RNA probe templateswere synthesized by introducing the T7 promoter into the antisensestrand and the SP6 promoter into the sense strand. The primer pair,AB5T7AS (5′-TAATACGACTCACTATAGGGATGTCTGGCTTTTTCCCTTCTTGAC-3′) (SEQ IDNO:15) and AB5SP6S (5′-GATTTAGGTGACACTATAGAAATTCAAGCTGGACGAATGACCCCA-3′)(SEQ ID NO:16), was used to generate the DNA template for antisense andsense RNA probes spanning 200 base pairs of human ABCB5 cDNA. Thissequence encodes ABCB5 amino acids 499-564 (GI:34539755). The primerpair CD133T7AS (5′-TAATACGACTCACTATAGGGAGCAGCCCCAGGACACAGCATA-3′) (SEQID NO:17) and CD133SP6S(5′-GATTTAGGTGACACTATAGAGACCCAAGACTCCCATAAAGC-3′) (SEQ ID NO:18) wasused to generate the DNA template for antisense and sense RNA probesspanning 200 base pairs of human CD133 cDNA, wherein this sequenceencodes CD133 amino acid 42-108 (GI: 5174386). The sequencespecificities for ABCB5 and CD133 were confirmed using the Genbankdatabase BLAST program. The RNA probes were labeled with digoxigenin(DIG) using the DIG RNA labeling kit (Roche Applied Science,Indianapolis, Ind.). For in situ hybridization, 8 μM frozen tissuesections were baked at 50° C. for 15 min, then fixed in 4%paraformaldehyde at room temperature (RT) for 20 min. The sections weretreated with 1 μg/ml proteinase K/PBS at 37° C. for 20 min andinactivated proteinase K with 0.2% glucine/PBS at RT for 5 min. Uponwashes with PBS 2×2 min, the tissue sections were fixed in 4%paraformadehyde at RT for 15 min, washed with PBS 2×5 min, and thentreated with 0.25% acetic anhydride/0.1M triethanolamine at RT for 10min. The tissue sections were placed in 2×SSC and then hybridized with500 ng/ml antisense or sense probe in hybridization buffer (0.3M NaCl,10 mM Tris-HCl pH 7.6, 5 mM EDTA, 1 × Denharts, 50% formamide, 100 μg/mltRNA and 10% dectran sulphate) at 42° C. overnight. Post hybridizationsections were treated with 0.2×SSC at 55° C. for 2×20 min, 20 □g/mlRNaseA in 0.5M NaCl, 10 mM Tris-HCl pH 7.5 at 37° C. for 30 min, and0.2×SSC at 55° C. for 20 min. The hybridized probes were immunodetectedusing the DIG detection kit (Roche) and the Tyramide SignalAmplification (TSA) kit (PerkinElmer, Boston, Mass.) as follows: 1×DIGblock buffer for 30 min, 1:100 anti-DIG Ab peroxidase conjugate at RTfor 1 hour, 1×DIG wash 3×5 min, TSA reagent 10 min at RT, PBS 2×5 min,1:100 streptavidin-horseradish peroxidase 30 min at RT, PBS 3×5 min. Thelabeling was visualized with NovaRed substrate (Vector Laboratories).

Animals.

BALB/c nude mice and NOD/SCID mice were purchased from The JacksonLaboratory (Bar Harbor, Me.). SCID mice (C.B-17) and BALB/c Rag2^(−/−)mice were purchased from Taconic (Germantown, N.Y.). The animals werehoused in autoclaved microisolator cages and were fed sterilized foodand water. Mice were maintained in accordance with the institutionalguidelines of Children's Hospital Boston and Harvard Medical School andexperiments were performed according to approved experimental protocols.

Human to Mouse Melanoma Xenotransplantation and Human to ChimericMouse/Human Skin Melanoma Xenotransplantation.

Human to mouse melanoma xenografts were established by subcutaneousinjection of human G3361, A375, SK-MEL-5 or clinical patient-derivedhuman melanoma cells in BALB/c nude or NOD/SCID mice as describedpreviously³. For human to chimeric mouse/human skin melanomaxenotransplantation, single donor-derived split human skin was obtainedin accordance with the Partners HealthCare Research ManagementInstitutional Review Board by cutting abdominal skin with a 0.016-inchgauge dermatome. Human skin was subsequently xenografted ontoimmunodeficient Rag2^(−/−) mice as described previously¹³, under aprotocol approved by the institutional animal committee. Briefly, twocircular graft beds, each, 1.5 cm² were prepared on bilateral dorsa of4-8 week old Rag2^(−/−) micetreated with antibiotics (1 tablet ofadditional food per week containing Amoxicillin (3 mg), Flagyl (0.69 mg)and Bismuth (0.185 mg)) to prevent Helicobacter pylorii infection. Humandonor skin was trimmed to conform to the bed and held in place withstaples until 10 days following surgery. Unsegregated or ABCB5⁺-depletedA375, MUM-2B or MUM-2C melanoma cells (2×10⁶ in 20 μl PBS) wereintradermally microinjected into grafts after 6 weeks stabilization. Allskin grafts were harvested in their entirety 3 weeks after tumour cellinoculation, fixed in formalin, serially sectioned and stained with H&Eusing standard methods for histological analysis of tumour formation.Sections representing maximum cross-sectional tumour area and thus bestapproximating the size of the generally spherical to ovoid tumournodules were evaluated. Tumour volume (TV) was histologically determinedand calculated as described³. Statistically significant differences inhistological tumour formation were assessed using the Fisher's Exacttest. Differences in tumour volume were statistically compared using thenonparametric Mann-Whitney Test, with a two-sided P value of P<0.05considered significant.

Stable Green Fluorescence Protein (GFP)-Transfected Melanoma Xenograftsto Human-SCID Chimeras.

Recombinant lentiviral vectors harboring GFP cDNA were obtained from Dr.M. Herlyn at the Wistar Institute and used to infect human A375 melanomacells by lentiviral gene transfer. Two days after infection, cells wereselected with puromycin (1 μg/ml) for a period of 7 days. Transgeneexpression was verified by fluorescence microscopy and flow cytometry.Melanoma xenografts were generated in human-SCID chimeras according tothe protocol previously described¹³. SCID mice (C.B-17) between 4-6weeks of age were purchased from Taconic (Germantown, N.Y.). Mice wereanesthetized and prepared for transplantation by shaving the hair from a2 cm² area on the dorsal torso followed by removal of full thicknessskin down to the fascia. Full thickness human foreskin grafts of thesame size were placed onto the wound beds. The skin grafts were thencovered by Vaseline-saturated gauze and secured with band aids and 3Msports tapes. After 10 days, the dressings were removed and the miceallowed to recover for approximately 4-5 weeks before melanomainoculation. GFP-labeled A375 melanoma cells were harvested andsuspended in PBS at a density of 10⁸ cells/ml. One hundred μl each ofcell suspension were injected intradermally into the human skin grafts.The tumour xenografts were then harvested in 3 weeks or when the tumourreaches 1 cm³ in size, and processed for frozen section. For doubleimmunofluorescence, frozen sections (5 μm thick) of melanoma xenograftswere fixed in 4% paraformaldehyde, blocked with donkey serum, andincubated sequentially with anti-CD144 (Cell Signaling, Danvers, Mass.),Texas red-conjugated donkey anti-rabbit (Invitrogen, Carlsbad, Calif.),anti-GFP (Novus Biologicals, Littleton, Colo.), and FITC-conjugateddonkey anti-goat Abs (Jackson ImmunoResearch, West Grove, Pa.). Afterwashing in PBS, the sections were coverslipped using an antiquenchmountant containing DAPI (VectaShield, Vector Laboratories, Burlingame,Calif.). Irrelevant isotype-matched primary Abs were included ascontrols.

Example 2

The mechanisms through which ABCB5⁺ MMIC or CSC in other cancers triggerand promote neoplastic progression are currently unknown. Wehypothesized that ABCB5⁺ MMIC possess vasculogenic differentiationplasticity and selectively drive melanoma growth through a specific rolein providing nutritional support to growing tumours based onpreferential co-expression in vivo of the vasculogenic differentiationmarkers CD144 (VE-cadherin) and TIE-1³ by the ABCB5⁺ tumourigenicminority population.

To further characterize the repertoire of genes differentially expressedin MMIC compared to tumour bulk populations, we first performedmicroarray analyses on purified ABCB5⁺ (n=5) and ABCB5⁻ (n=5) cellsubsets derived from the established human melanoma cell lines G3361 andA375 and from three separate patient-derived melanoma specimens, allpreviously characterized in our laboratory with regard to ABCB5expression and MMIC phenotype in human melanoma xenotransplantationassays³. Using this approach¹⁰, 399 genes were identified that weredifferentially expressed (P<0.05) between ABCB5⁺ MMIC and ABCB5⁻melanoma bulk populations (Table 5), in addition to ABCB5 shownoverexpressed in ABCB5⁺ purified populations by real-time PCR (P<0.05).One identified functional gene network, validated by PCR-based geneexpression analyses in ABCB5⁺ melanoma cell subsets, showed keymolecules of vasculogenesis (the ability to differentiate alongendothelial lines), FLT1 (VEGFR-1) and PTK2 (FAK), and of angiogenesis(the ability to induce ingrowth and proliferation of mature stromalblood vessels), FLT1 (VEGFR-1), PTK2 (FAK), MET (HGFR), NRP2, and ETS1,to be specifically overexpressed in ABCB5⁺ MMIC (FIG. 1a,b ).

Another set of genes differentially expressed in ABCB+ melanoma stemcells vs. ABCB5-melanoma bulk population cells was identified usingRT-PCR. The data is shown in Table 6. The fold-expression levels areshown in the 7^(th) column and can be compared to the control valuesshown in the last few rows of the table. A positive value indicates thatthe gene had higher expression levels in ABCB5+ cells and a negativevalue indicates that the gene had higher expression levels in ABCB5−cells. Some of the genes exhibited a greater than 100-fold and some evengreater 1000-fold increase in expression in ABCB5+ versus ABCB5− cells.The highly expressed genes include factors that are likely secreted bythe stem cells which may act on cells in a tumor either in an autocrinefashion on tumor stem cells, or in a paracrine fashion also on bulkpopulation cancer cells. Appropriate therapies can be designed to treatcancers by inhibiting the expression or activity of such factors.

Preferential expression of VEGFR-1 by ABCB5⁺ MMIC vs. ABCB5⁻subpopulations was also demonstrated by dual-color flow cytometry at theprotein level (15.6±5.3% vs. 4.4±2.0% of cells, respectively,mean±s.e.m., n=6, P<0.05) (FIG. 1c ). To determine whether VEGF/VEGFR-1interaction in MMIC influenced expression of the vasculogenicdifferentiation marker CD144, we evaluated functionally the effects ofVEGF signaling in purified ABCB5⁺ MMIC or ABCB5⁻ melanomasubpopulations. VEGF (100 ng/ml¹¹) selectively induced expression ofCD144 at high levels in VEGFR-1-expressing ABCB5⁺ but notVEGFR-1-negative ABCB5⁻ melanoma cells, to 36.2±5.7% vs. 4.8±2.7% ofcells (mean±s.e.m., n=3), respectively (P<0.01) (FIG. 1d ).Preincubation with a blocking monoclonal antibody (mAb) to VEGFR-1abrogated the ability of VEGF to induce CD144 expression in humanmelanoma cells (0.0±0.0% in VEGFR-1 mAb-treated vs. 64±1% or 57±3% inuntreated or isotype control mAb-treated cultures, respectively,mean±s.e.m., n=3, P<0.01) (FIG. 1e ). Moreover, VEGFR-1 mAb stronglyinhibited VEGF-induced multicellular capillary-like tube formation byhuman melanoma cells in established in vitro vasculogenicdifferentiation assays¹¹, with significantly reduced numbers of tubesformed/microscopy field (6.7±0.9 in VEGFR-1 mAb-treated vs. 99.0±24.0 or76.7±3.3% in untreated or isotype control mAb-treated cultures,respectively, mean±s.e.m., n=3, P<0.05), and significantly lower averagetube length (33.2±4.5 μm in VEGFR-1 mAb-treated vs. 92.1±1.6 μm or86.5±1.7 μm in untreated or isotype control mAb-treated cultures,respectively, mean±s.e.m., n=3, P<0.001) (FIG. 1f ). In contrast, bothABCB5⁺ MMIC and ABCB5⁻ melanoma bulk population exhibited similaradipogenic and osteogenic differentiation capacity previously detectedin human melanoma cells¹² (adipogenesis: 100.0±0.0% vs. 93.2±6.9% ofcells Oil Red-positive, respectively; mean±s.e.m., n=3, NS;osteogenesis: 90.8±9.2% vs. 98.3±1.7% of cells Alizarin Red-positive,respectively; mean±s.e.m., n=3, NS) (FIG. 1g,h ), and neither ABCB5⁺MMIC nor ABCB5⁻ melanoma bulk population exhibited capacity for myogenicdifferentiation¹⁰ (0.0±0.0% vs. 0.0±0.0% of cells myogenin-positive,respectively; mean±s.e.m., n=3, NS) (FIG. 1h ). The selective in vitrovasculogenic differentiation capacity of ABCB5⁺ MMIC in response toVEGF/VEGFR-1 signaling indicated a potential role of this CSC subset intumour vasculogenesis.

TABLE 5 Differentially expressed genes between ABCB5⁺ MMIC and ABCB5⁻melanoma bulk populations (P < 0.05). Molecules ID Fold Change AABHD7239579_at 0.661 ACBD6 225317_at 0.83 AK3 224655_at 0.845 AKAP9 215483_at2.168 AKR1CL2 1559982_s_at 1.732 AMZ2 227567_at 1.377 ANAPC5 235926_at2.631 ANK2 202921_s_at 4.162 ANKH 229176_at 0.776 ANKRD28 241063_at2.297 ANKRD44 226641_at 1.218 ANKRD52 228257_at 0.762 ANXA4 201302_at0.83 AOC3 204894_s_at 1.894 APBB2 40148_at 1.139 ARS2 201679_at 1.307ASCC3L1 214982_at 3.009 (includes EG:23020) ASPM 232238_at 1.411 ATAD2235266_at 1.304 ATP5I 207335_x_at 0.737 ATXN2L 207798_s_at 1.656 BARD1205345_at 1.559 BAT3 230513_at 0.697 BCL9L 227616_at 1.291 BDP1224227_s_at 1.632 BLID 239672_at 1.91 BRI3 223376_s_at 0.792 BUB1216277_at 1.856 (includes EG:699) BUB1 233445_at 3.209 (includes EG:699)C10ORF18 244165_at 2.046 C12ORF45 226349_at 0.688 C12ORF48 220060_s_at1.216 C12ORF51 230216_at 2.874 C12ORF51 1557529_at 3.632 C14ORF1351563259_at 1.353 C14ORF156 221434_s_at 0.867 C16ORF63 225087_at 0.872C18ORF10 213617_s_at 0.754 C18ORF10 212055_at 0.737 C19ORF42 219097_x_at0.813 C20ORF4 234654_at 1.731 C22ORF28 200042_at 0.829 C22ORF30216555_at 1.521 C2ORF30 224630_at 0.851 C5ORF24 229098_s_at 1.531C9ORF78 218116_at 0.789 C9ORF85 244160_at 1.52 CABIN1 1557581_x_at 3.052CAMK2D 225019_at 0.823 CAMK2D 228555_at 0.758 CANX 238034_at 0.8 CAPZB201949_x_at 0.764 CASC5 228323_at 1.144 CBS 240517_at 1.818 CCDC127226515_at 0.835 CCDC14 240884_at 1.771 CCDC52 234995_at 1.166 CCDC57214818_at 1.703 CCDC73 239848_at 1.294 CCDC93 219774_at 1.208 CDC14B234605_at 2.512 CDC16 242359_at 6.261 CENPJ 234023_s_at 1.22 CENPJ220885_s_at 1.64 CEP27 228744_at 0.651 CEP55 218542_at 1.096 CGGBP1224600_at 0.913 CHD2 244443_at 1.757 CHD8 212571_at 1.27 CLN8 229958_at1.344 CNIH3 232758_s_at 1.451 COBRA1 1556434_at 1.985 COIL 203653_s_at1.259 COL4A2 211966_at 0.729 COQ4 218328_at 1.328 CPEB2 226939_at 1.251CPNE3 202119_s_at 0.833 CREB1 204313_s_at 0.791 CREB3L2 237952_at 2.013CRIPAK 228318_s_at 1.486 CROP 242389_at 2.121 CSE1L 201112_s_at 0.911CSE1L 210766_s_at 0.885 CUL4A 232466_at 2.607 CYB5R3 1554574_a_at 0.793DARS 201624_at 0.928 DCLRE1C 242927_at 1.187 DCUN1D2 240478_at 1.76DDX17 213998_s_at 1.528 DDX52 212834_at 0.771 DEGS1 209250_at 0.804DEPDC1 232278_s_at 1.119 DHX40 218277_s_at 0.812 DNAJC21 230893_at 0.829DNM1L 236032_at 1.503 DTX3 49051_g_at 1.32 ECHDC1 233124_s_at 0.943EIF2S1 201142_at 0.717 EIF2S1 201144_s_at 0.824 EIF4G3 201935_s_at 1.174ELOVL2 213712_at 0.699 EMP2 225079_at 0.781 ENAH 222433_at 0.783 ENDOD1212573_at 0.775 ENTPD5 231676_s_at 0.867 ERBB3 1563253_s_at 0.691 ERRFI1224657_at 0.881 ETS1 241435_at 1.797 EWSR1 229966_at 1.686 EXT1242126_at 2.116 FAM62C 239770_at 1.551 FAM98A 212333_at 0.767 FHL3218818_at 0.546 FLJ10357 241627_x_at 2.31 FLJ31306 239432_at 1.753 FLT1232809_s_at 1.861 FOXN3 218031_s_at 0.721 FUBP1 240307_at 2.087GABARAPL2 209046_s_at 0.863 GABPA 243498_at 2.03 GALNT1 201722_s_at0.926 GBF1 233114_at 2.03 GGT1 211417_x_at 1.555 GHITM 1554510_s_at0.764 GMFB 202544_at 0.904 GNPDA1 202382_s_at 0.787 GOLGA8A 213650_at2.289 GPD2 243598_at 2.13 GPR107 211979_at 0.843 GPR135 241085_at 1.851HDAC3 240482_at 2.062 HEATR2 241352_at 0.784 HECW1 237295_at 11.843HELLS 242890_at 1.359 HERC5 219863_at 1.156 HIAT1 225222_at 0.832 HNRNPC235500_at 1.769 HNRPD 235999_at 1.92 HNRPD 241702_at 1.962 HNRPH1213472_at 2.332 HOXA2 228642_at 1.44 HOXB9 216417_x_at 0.766 HOXD3206601_s_at 1.897 HPS1 239382_at 1.749 HSD17B1 228595_at 0.753 HSDL2209513_s_at 0.803 HSPA4L 205543_at 0.786 HUWE1 214673_s_at 1.858 IDS1559136_s_at 2.001 IFNGR1 242903_at 2.171 IGHMBP2 215980_s_at 0.893IL13RA1 201887_at 0.775 INSIG2 209566_at 0.872 IPO7 200993_at 0.875 IPW213447_at 1.399 IRS2 236338_at 2.162 JARID1A 226367_at 1.192 JARID2232835_at 2.139 KIAA0841 36888_at 1.389 KIAA0907 230028_at 1.83 KIAA1267224489_at 1.355 KIAA1618 231956_at 2.27 KIAA1737 225623_at 0.837KIAA2013 1555933_at 2.18 KIDINS220 1557246_at 2.97 KPNA6 226976_at 0.814KRTAP19-1 1556410_a_at 2.07 KSR2 230551_at 3.211 LBA1 213261_at 1.225LIMS1 212687_at 0.822 LOC126917 225615_at 0.819 LOC137886 212934_at0.886 LOC145757 1558649_at 2.779 LOC145786 229178_at 1.907 LOC1463251553826_a_at 3.943 LOC203547 225556_at 0.802 LOC219731 1557208_at 0.419LOC254128 1557059_at 2.164 LOC283888 1559443_s_at 2.56 LOC285147236166_at 2.377 LOC338799 226369_at 1.137 LOC388135 230475_at 1.979LOC388969 232145_at 1.555 LOC389203 225014_at 0.79 LOC641298 208118_x_at1.419 LOC645166 228158_at 0.823 LOC645513 239556_at 2.24 LOC729397236899_at 2.231 LRCH3 229387_at 1.793 LRRFIP1 239379_at 1.796 MAEA207922_s_at 0.765 MALAT1 224568_x_at 1.699 MALAT1 223940_x_at 1.659MAP1LC3B 208785_s_at 0.808 MAP2K4 203266_s_at 0.881 MAP3K15 200979_at0.741 6-Mar 201737_s_at 1.219 MBNL1 201152_s_at 0.867 MDM4 235589_s_at1.629 MECR 218664_at 0.832 MED19 226300_at 0.782 MEF2C 236395_at 2.104MET 213816_s_at 1.283 MIA3 1569057_s_at 0.759 MLL 212079_s_at 1.599MOBKL1B 214812_s_at 0.762 MRPL42 217919_s_at 0.866 (includes EG:28977)MRPL51 224334_s_at 0.846 MTERFD3 225341_at 1.422 MTUS1 239576_at 1.975MYO10 243159_x_at 2.528 MYO10 244350_at 1.677 N4BP2L1 213375_s_at 2.01N4BP2L2 235547_at 1.631 N4BP2L2 242576_x_at 2.349 NAALAD2 1554506_x_at0.464 NANP 228073_at 0.817 NAPA 239362_at 1.624 NAPE-PLD 242635_s_at1.216 NARG1 1556381_at 2.827 NAT8B 206964_at 2.513 NBPF16 201104_x_at1.411 NBR1 1568856_at 1.957 NCKAP1L 209734_at 2.071 NDFIP1 217800_s_at0.815 NDUFAF2 228355_s_at 0.722 NDUFB6 203613_s_at 0.712 NEK1 213328_at1.381 NFATC2IP 217527_s_at 1.272 NPAS2 1557690_x_at 1.76 NPTN 228723_at2.086 NRP2 210841_s_at 1.106 NUCB2 203675_at 0.812 NUDT4 212183_at 0.685NUPL1 241425_at 2.179 OCIAD1 235537_at 1.794 ORMDL1 223187_s_at 1.171OSBPL5 233734_s_at 1.261 OSGEP 242930_at 1.541 PABPN1 213046_at 2.228PAK1 226507_at 0.869 PAPD4 222282_at 3.39 PDE4B 215671_at 3.457 PDHB211023_at 0.827 PDHB 208911_s_at 0.807 PDK1 239798_at 1.654 PDLIM5212412_at 0.752 PDSS1 236298_at 1.64 PDXDC1 1560014_s_at 2.105 PGRMC2213227_at 0.686 PHC1 218338_at 1.123 PHF20L1 219606_at 2.3 PIGY224660_at 0.793 (includes EG:84992) PIP5K3 1557719_at 2.227 PITPNA201190_s_at 0.863 PMP22 210139_s_at 0.865 PMS2L3 214473_x_at 1.159POFUT2 207448_at 1.759 POLR2J2 1552622_s_at 1.828 POLR2J2 1552621_at1.652 POP4 202868_s_at 0.847 PPP1R3D 204554_at 0.805 PPP1R7 201213_at0.698 PPP3CA 202457_s_at 0.867 PRO1073 228582_x_at 1.607 PRPF38B230270_at 1.888 PSEN1 242875_at 1.851 PSMA2 201316_at 0.839 PSMA3201532_at 0.798 PTK2 234211_at 2.539 PTPMT1 229535_at 0.769 RAB11FIP3228613_at 2.546 RAB11FIP3 216043_x_at 0.551 RAB14 200927_s_at 0.772RAB1A 213440_at 0.81 RAD54L 204558_at 1.483 RADIL 223693_s_at 2.126RBM25 1557081_at 1.57 RBM26 229433_at 1.43 RBM4 213718_at 1.53 RBM5209936_at 2.249 RFT1 240281_at 1.426 RHOA 240337_at 2.143 RHOBTB21556645_s_at 1.538 RLBP1L1 224996_at 0.835 RNF43 228826_at 1.401RP11-139H14.4 1569124_at 11.472 RPE 221770_at 0.766 RPE 225039_at 0.787RPL7L1 225515_s_at 0.899 RUNX3 204198_s_at 1.233 SDAD1 242190_at 3.009SDCCAG8 243963_at 2.67 SEC16B 1552880_at 1.877 SEPHS1 208940_at 0.87511-Sep 201307_at 0.784 SF1 210172_at 2.452 SF3B1 201070_x_at 1.35 SF3B1214305_s_at 1.359 SFRS15 222311_s_at 1.818 SFRS15 243759_at 2.028 SGCA1562729_at 2.395 SGOL2 235425_at 1.591 SH2B3 203320_at 0.806 SKP1200718_s_at 0.898 SLC16A1 202235_at 0.83 SLC20A1 230494_at 1.884 SLC2A11232167_at 1.529 SLC2A8 239426_at 2.012 SLC30A9 237051_at 2.063 SMA4238446_at 2.035 SMC6 218781_at 1.203 SMYD2 212922_s_at 0.867 SNORA28241843_at 1.628 SNRPA1 242146_at 3.54 SON 201085_s_at 1.144 SPOPL225659_at 0.828 SQLE 213577_at 1.502 SRP72 208801_at 0.751 SRP72208803_s_at 0.766 SRPRB 218140_x_at 0.767 STK36 234005_x_at 1.335 STK36231806_s_at 1.362 STRAP 1558002_at 2.189 STX11 235670_at 0.778 STX8204690_at 0.819 SUPT7L 201838_s_at 0.865 SVIL 215279_at 2.228 SYNE2202761_s_at 1.356 TAF15 227891_s_at 1.971 TAF1B 239046_at 1.468 TAOK3220761_s_at 1.195 TBC1D5 201814_at 0.782 TBC1D8 221592_at 1.246 TBC1D8204526_s_at 1.373 TBXA2R 207554_x_at 0.877 TBXA2R 336_at 0.73 TCAG7.907238678_at 1.546 TCOF1 202385_s_at 1.169 (includes EG:6949) TFB1M228075_x_at 0.87 THRAP3 217847_s_at 1.464 TIMM23 218119_at 0.723 TM6SF11558102_at 0.704 TMEM126B 221622_s_at 0.843 TMEM165 1560622_at 1.756TMEM30A 232591_s_at 0.771 TNFAIP1 201207_at 0.88 TNPO1 1556116_s_at1.739 TNRC6A 234734_s_at 1.268 TOX4 201685_s_at 0.73 TPM4 235094_at2.079 TRAPPC2 219351_at 0.821 TRAPPC2L 218354_at 0.837 TRIM33 239716_at2.496 TRIM46 238147_at 1.96 TRIO 240773_at 2.607 TRNT1 243236_at 2.295TRPV1 1556229_at 2.636 TSPAN31 203227_s_at 0.744 TTC26 233999_s_at 1.184TTC3 208664_s_at 1.396 TTC9C 1569189_at 1.55 TTLL4 1557611_at 2.092TXNDC12 223017_at 0.849 TXNL1 243664_at 1.98 UBE2E3 210024_s_at 0.758UBE3C 1560739_a_at 0.815 UBXD7 212840_at 0.754 UGT1A6 206094_x_at 3.86UNK 1562434_at 1.637 UQCC 229672_at 1.451 USP36 224979_s_at 1.393 USP8229501_s_at 0.808 VPS37B 236889_at 2.85 VTI1B 209452_s_at 0.821 WDR41218055_s_at 0.789 WDR68 233782_at 1.924 WFS1 1555270_a_at 1.315 WIPF2216006_at 2.916 WTAP 1560274_at 1.747 XRCC5 232633_at 2.106 YY1224711_at 0.821 ZFHX3 215828_at 1.737 ZFR 238970_at 2.655 ZFX207920_x_at 1.625 ZMYND8 209049_s_at 1.102 ZNF154 242170_at 2.667 ZNF224216983_s_at 2.986 ZNF226 219603_s_at 1.332 ZNF251 226754_at 1.313 ZNF292236435_at 3.201 ZNF326 241720_at 1.418 ZNF337 1565614_at 2.096 ZNF536233890_at 3.303 ZNF567 242429_at 2.103 ZNF618 226590_at 0.75 ZNF668219047_s_at 0.691 ZNF800 227101_at 1.484 ZUFSP 228330_at 1.205

TABLE 6 Differentially expressed genes between ABCB5⁺ and ABCB5⁻ cellsas detected by RT-PCR. PCR Array Catalog #: PAHS-024 ABCB5+/ ABCB5− FoldPosition Unigene Refseq Symbol Description Gname change A01 Hs.525622NM_005163 AKT1 V-akt murine thymoma AKT/PKB 1.2687 viral oncogenehomolog 1 A02 Hs.369675 NM_001146 ANGPT1 Angiopoietin 1 AGP1/AGPT 1.2953A03 Hs.583870 NM_001147 ANGPT2 Angiopoietin 2 AGPT2/ANG2 2.7007 A04Hs.209153 NM_014495 ANGPTL3 Angiopoietin-like 3 ANGPT5 3.0596 A05Hs.9613 NM_001039667 ANGPTL4 Angiopoietin-like 4 ANGPTL2/ARP4 1.6974 A06Hs.1239 NM_001150 ANPEP Alanyl (membrane) APN/CD13 1.3597 aminopeptidase(aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, CD13,p150) A07 Hs.194654 NM_001702 BAI1 Brain-specific FLJ41988 3.0596angiogenesis inhibitor 1 A08 Hs.54460 NM_002986 CCL11 Chemokine (C-Cmotif) SCYA11 1.8834 ligand 11 A09 Hs.303649 NM_002982 CCL2 Chemokine(C-C motif) GDCF-2/ 2.0326 ligand 2 GDCF-2HC11 A10 Hs.76206 NM_001795CDH5 Cadherin 5, type 2, VE- 7B4/CD144 3.0596 cadherin (vascularepithelium) A11 Hs.517356 NM_030582 COL18A1 Collagen, type XVIII, KNO1.9634 alpha 1 A12 Hs.570065 NM_000091 COL4A3 Collagen, type IV, alphaTUMSTATIN 2.1634 3 (Goodpasture antigen) B01 Hs.789 NM_001511 CXCL1Chemokine (C-X-C FSP/GRO1 1.2086 motif) ligand 1 (melanoma growthstimulating activity, alpha) B02 Hs.632586 NM_001565 CXCL10 Chemokine(C-X-C C7/IFI10 −2.1987 motif) ligand 10 B03 Hs.89690 NM_002090 CXCL3Chemokine (C-X-C CINC-2b/ 2.061 motif) ligand 3 GRO3 B04 Hs.89714NM_002994 CXCL5 Chemokine (C-X-C ENA-78/ 1.8834 motif) ligand 5 SCYB5B05 Hs.164021 NM_002993 CXCL6 Chemokine (C-X-C CKA-3/ 2.1936 motif)ligand 6 GCP-2 (granulocyte chemotactic protein 2) B06 Hs.77367NM_002416 CXCL9 Chemokine (C-X-C CMK/Humig −1.1225 motif) ligand 9 B07Hs.592212 NM_001953 TYMP Thymidine ECGF1/ 1.5837 phosphorylase MNGIE B08Hs.154210 NM_001400 EDG1 Endothelial CHEDG1/ 1.0377 differentiation,D1S3362 sphingolipid G-protein- coupled receptor, 1 B09 Hs.516664NM_182685 EFNA1 Ephrin-A1 B61/ECKLG 1.4573 B10 Hs.516656 NM_004952 EFNA3Ephrin-A3 EFL2/EPLG3 1.3692 B11 Hs.149239 NM_004093 EFNB2 Ephrin-B2EPLG5/HTKL 1.1355 B12 Hs.419815 NM_001963 EGF Epidermal growth factorHOMG4/URG 187.8365 (beta-urogastrone) C01 Hs.76753 NM_000118 ENGEndoglin (Osler-Rendu- CD105/END 1.1514 Weber syndrome 1) C02 Hs.437008NM_004444 EPHB4 EPH receptor B4 HTK/MYK1 1.3692 C03 Hs.115263 NM_001432EREG Epiregulin ER 1.8834 C04 Hs.483635 NM_000800 FGF1 Fibroblast growthfactor AFGF/ECGF 1.5511 1 (acidic) C05 Hs.284244 NM_002006 FGF2Fibroblast growth factor BFGF/FGFB 1.1355 2 (basic) C06 Hs.1420NM_000142 FGFR3 Fibroblast growth factor ACH/CD333 1.7092 receptor 3(achondroplasia, thanatophoric dwarfism) C07 Hs.11392 NM_004469 FIGFC-fos induced growth VEGF-D/VEGFD 3.5884 factor (vascular endothelialgrowth factor D) C08 Hs.654360 NM_002019 FLT1 Fms-related tyrosineFLT/VEGFR1 2.4172 kinase 1 (vascular endothelial growth factor/vascularpermeability factor receptor) C09 Hs.388245 NM_021973 HAND2 Heart andneural crest DHAND2/Hed 2.0186 derivatives expressed 2 C10 Hs.396530NM_000601 HGF Hepatocyte growth factor F-TCF/HGFB 4.542 (hepapoietin A;scatter factor) C11 Hs.654600 NM_001530 HIF1A Hypoxia-inducible factorHIF-1alpha/ −1.0918 1, alpha subunit (basic HIF1 helix-loop-helixtranscription factor) C12 Hs.44227 NM_006665 HPSE Heparanase HPA/HPR1286.6871 D01 Hs.504609 NM_002165 ID1 Inhibitor of DNA binding ID -1.03291, dominant negative helix-loop-helix protein D02 Hs.76884 NM_002167 ID3Inhibitor of DNA binding HEIR-1 −1.3535 3, dominant negativehelix-loop-helix protein D03 Hs.37026 NM_024013 IFNA1 Interferon, alpha1 IFL/IFN 1.8834 D04 Hs.93177 NM_002176 IFNB1 Interferon, beta 1,IFB/IFF 1.8834 fibroblast D05 Hs.856 NM_000619 IFNG Interferon, gammaIFG/IFI 1.8834 D06 Hs.160562 NM_000618 IGF1 Insulin-like growth factorIGFI 4.7022 1 (somatomedin C) D07 Hs.126256 NM_000576 IL1B Interleukin1, beta IL-1/IL1-BETA 2.0898 D08 Hs.654458 NM_000600 IL6 Interleukin 6(interferon, BSF2/HGF 1.7331 beta 2) D09 Hs.624 NM_000584 IL8Interleukin 8 3-10C/AMCF-I 1.1674 D10 Hs.436873 NM_002210 ITGAVIntegrin, alpha V CD51/DKFZ 1.217 (vitronectin receptor, p686A08142alpha polypeptide, antigen CD51) D11 Hs.218040 NM_000212 ITGB3 Integrin,beta 3 (platelet CD61/GP3A −1.0619 glycoprotein IIIa, antigen CD61) D12Hs.224012 NM_000214 JAG1 Jagged 1 (Alagille AGS/AHD 1566.5046 syndrome)E01 Hs.479756 NM_002253 KDR Kinase insert domain CD309/FLK1 1.234receptor (a type III receptor tyrosine kinase) E02 Hs.473256 NM_005560LAMA5 Laminin, alpha 5 KIAA1907 3.8727 E03 Hs.421391 NM_007015 LECT1Leukocyte cell derived BRICD3/ 1.8834 chemotaxin 1 CHM-I E04 Hs.194236NM_000230 LEP Leptin OB/OBS 2.1485 E05 Hs.82045 NM_002391 MDK Midkine(neurite growth- MK/NEGF2 1.4573 promoting factor 2) E06 Hs.513617NM_004530 MMP2 Matrix metallopeptidase CLG4/CLG4A 1.674 2 (gelatinase A,72 kDa gelatinase, 72 kDa type IV collagenase) E07 Hs.297413 NM_004994MMP9 Matrix metallopeptidase CLG4B/GELB 1.9097 9 (gelatinase B, 92 kDagelatinase, 92 kDa type IV collagenase) E08 Hs.436100 NM_004557 NOTCH4Notch homolog 4 INT3/NOTCH3 1.2002 (Drosophila) E09 Hs.131704 NM_003873NRP1 Neuropilin 1 CD304/ 1.1755 DKFZp686A03134 E10 Hs.471200 NM_003872NRP2 Neuropilin 2 NP2/NPN2 1.4373 E11 Hs.707991 NM_002607 PDGFAPlatelet-derived growth PDGF-A/ 1.2002 factor alpha polypeptide PDGF1E12 Hs.514412 NM_000442 PECAM1 Platelet/endothelial cell CD31/PECAM-111.9037 adhesion molecule (CD31 antigen) F01 Hs.81564 NM_002619 PF4Platelet factor 4 CXCL4/ 2.9966 (chemokine (C-X-C SCYB4 motif) ligand 4)F02 Hs.252820 NM_002632 PGF Placental growth factor, D12S1900/PGFL−1.1865 vascular endothelial growth factor-related protein F03 Hs.77274NM_002658 PLAU Plasminogen activator, ATF/UPA 1.6396 urokinase F04Hs.143436 NM_000301 PLG Plasminogen DKFZp779M0222 1.8834 F05 Hs.125036NM_020405 PLXDC1 Plexin domain containing DKFZp686F0937/ 3.4184 1 TEM3F06 Hs.528665 NM_021935 PROK2 Prokineticin 2 BV8/KAL4 1.8446 F07Hs.201978 NM_000962 PTGS1 Prostaglandin- COX1/COX3 1.2086 endoperoxidesynthase 1 (prostaglandin G/H synthase and cyclooxygenase) F08 Hs.532768NM_002615 SERPINF1 Serpin peptidase EPC-1/PEDF 1.1121 inhibitor, clade F(alpha- 2 antiplasmin, pigment epithelium derived factor), member 1 F09Hs.68061 NM_021972 SPHK1 Sphingosine kinase 1 SPHK 1.192 F10 Hs.301989NM_015136 STAB1 Stabilin 1 CLEVER-1/FEEL-1 4.357 F11 Hs.89640 NM_000459TEK TEK tyrosine kinase, CD202B/TIE-2 −1.2805 endothelial (venousmalformations, multiple cutaneous and mucosal) F12 Hs.170009 NM_003236TGFA Transforming growth TFGA 3549.3357 factor, alpha G01 Hs.645227NM_000660 TGFB1 Transforming growth CED/DPD1 1.1837 factor, beta 1 G02Hs.133379 NM_003238 TGFB2 Transforming growth TGF-beta2 1.3787 factor,beta 2 G03 Hs.494622 NM_004612 TGFBR1 Transforming growth AAT5/ACVRLK41.7695 factor, beta receptor I (activin A receptor type II-like kinase,53 kDa) G04 Hs.164226 NM_003246 THBS1 Thrombospondin 1 THBS/TSP −1.0619G05 Hs.371147 NM_003247 THBS2 Thrombospondin 2 TSP2 −1.203 G06 Hs.522632NM_003254 TIMP1 TIMP metallopeptidase CLGI/EPA −1.1147 inhibitor 1 G07Hs.633514 NM_003255 TIMP2 TIMP metallopeptidase CSC-21K 1.2864 inhibitor2 G08 Hs.701968 NM_000362 TIMP3 TIMP metallopeptidase HSMRK222/ 1.8834inhibitor 3 (Sorsby K222 fundus dystrophy, pseudoinflammatory) G09Hs.241570 NM_000594 TNF Tumor necrosis factor DIF/TNF-alpha 4.0652 (TNFsuperfamily, member 2) G10 Hs.525607 NM_006291 TNFAIP2 Tumor necrosisfactor, B94 1.8834 alpha-induced protein 2 G11 Hs.73793 NM_003376 VEGFAVascular endothelial VEGF/VEGF-A 2.4509 growth factor A G12 Hs.435215NM_005429 VEGFC Vascular endothelial Flt4-L/VRP 446.7529 growth factor CH01 Hs.534255 NM_004048 B2M Beta-2-microglobulin B2M −1.2983 H02Hs.412707 NM_000194 HPRT1 Hypoxanthine HGPRT/HPRT −1.2894phosphoribosyltransferase 1 (Lesch-Nyhan syndrome) H03 Hs.523185NM_012423 RPL13A Ribosomal protein L13a RPL13A 1.1837 H04 Hs.544577NM_002046 GAPDH Glyceraldehyde-3- G3PD/GAPD −1.146 phosphatedehydrogenase H05 Hs.520640 NM_001101 ACTB Actin, beta PS1TP5BP1 −1.0329H06 N/A SA_00105 HGDC Human Genomic DNA HIGX1A 1.8834 Contamination H07N/A SA_00104 RTC Reverse Transcription RTC 1.7451 Control H08 N/ASA_00104 RTC Reverse Transcription RTC 1.7451 Control H09 N/A SA_00104RTC Reverse Transcription RTC 1.7695 Control H10 N/A SA_00103 PPCPositive PCR Control PPC 1.8067 H11 N/A SA_00103 PPC Positive PCRControl PPC 1.7818 H12 N/A SA_00103 PPC Positive PCR Control PPC36695.9527

Example 3

We therefore tested the hypothesis that MMIC, as defined by the novelmarker ABCB5³, specifically relate to the phenomenon of vasculogenicmimicry whereby melanoma cells form channels capable of conductingnutrients from peripheral blood and thus serving as surrogates formature tumour vessels⁴. Because we posited that this phenomenon may bemore robust during early stages of tumour formation before cancerangiogenesis fully develops, we evaluated experimentally-inducedhuman-derived melanomas grown as tumour xenografts in the subcutis ofimmunodeficient mice and in the dermis of human skin xenografted toimmunodeficient mice (FIG. 2), the latter a humanized model wherebyhuman melanoma develops in the context of the human stromalmicroenvironment¹³. While only peripheral tumour vessels expressed themature endothelial marker, CD31, more interior regions of the melanomasexhibited formation of CD31-negative anastomosing channels withhistologic, histochemical (PAS-D and laminin reactivity), andultrastructural findings consistent with established features ofvasculogenic mimicry⁴ (FIG. 2a-d ). By electron microscopy, lumen-likespaces in these regions were lined by basement membrane-like materialand viable melanoma cells and contained erythrocytes surrounded byfinely granular matrix consistent with plasma, suggesting communicationwith the systemic circulation⁴. By immunohistochemistry and in situhybridization, channels expressed ABCB5 protein and mRNA, respectively(FIG. 2f-h ), which also correlated in vitro when assayed across a panelof human melanoma cell lines (FIG. 4), and the architecture of ABCB5⁺channels in xenografted tumours was identical to that focally detectedin patient-derived melanomas (FIG. 2f , inset). An identical pattern wasalso observed for CD133 mRNA (not illustrated), an additional marker fortumourigenic melanoma cells¹⁴ and melanoma progression¹⁵. Anti-ABCB5 mAbsystemically administered in vivo localized to channels, furtherconfirming their systemic perfusion as well as the intimate associationof ABCB5⁺ melanoma cells with channel lumens (FIG. 2i ). Double-labelingdemonstrated co-localization of the human endothelial markers CD144 andTIE-1 with ABCB5⁺ cells forming channels (FIG. 2j,k ). Tumours initiatedby melanoma cell lines expressing the green fluorescence protein (GFP)transgene confirmed the presence of melanoma cells lining channels thatco-expressed melanoma-associated GFP and CD144 (FIG. 2e ), as well ashuman melanoma—but not human xenograft-associated class I majorhistocompatibility complex (MHC) antigens (not illustrated). These datashow that the formation of perfused vessel-like channels in humanmelanoma is mediated by the ABCB5⁺ MMIC subpopulation found toselectively display gene profiles and differentiation capacityconsistent with its participation in tumour vasculogenesis.

Example 4

We next reasoned that if vasculogenic channel formation mediated byABCB5⁺ MMIC was functionally required for their capacity to efficientlyinitiate and drive melanoma growth, depletion of MMIC to low levelsshould inhibit the melanoma-associated vasculogenic response. Toevaluate tumourigenesis and vasculogenesis in a bioassay most relevantto human primary melanoma, we again utilized a human skin/murinexenograft model whereby melanomas develop in the relevant dermalmicroenvironment of human skin and express architectural features andevolutionary growth patterns more akin to naturally occurring lesions¹³.Intradermal orthotopic transplantation of 2×10⁶ unsegregated A375cutaneous melanoma cells (ABCB5 positivity: 5.2±5.1%; mean±s.e.m., n=9)(FIG. 3a ) or heterotopic transplantation of 2×10⁶ unsegregated uvealmelanoma cells previously assayed for vasculogenicdifferentiation^(4,16) (MUM-2B and MUM-2C, ABCB5 positivity: 2.46±0.46%and 3.81±1.04%, respectively; mean±s.e.m., n=3-4) (FIG. 3a ) to humanskin resulted in tumour formation three weeks following microinjectionsin 14 of 14 recipient skin grafts (A375: n=6, MUM-2B: n=4, MUM-2C: n=4replicates) when assessed histologically in serial sections of eachhuman skin xenograft in its entirety (FIG. 3b,c ). In contrast,intradermal transplantation of equal numbers of ABCB5⁺-depleted melanomacells resulted in histologically-assessed tumour formation in only 6 of14 recipient skin grafts (P<0.002) (FIG. 3b,c ) andhistologically-determined mean tumour volumes (TV) were significantlyreduced in recipients of ABCB5⁺-depleted vs. unsegregated melanomainocula (TV=2.8±1.8 mm³ vs. 10.9±6.9 mm³, respectively; mean±s.e.m.,P<0.005) (FIG. 3d ). When vasculogenic channel formation within tumourswas evaluated using quantitative image analysis technology to assess thepixilated density of laminin immunoreactivity, significantly fewerchannels per cross-sectional area were detected in tumours that formedfrom MMIC-depleted inocula compared to those that originated fromunsegregated tumour cell grafts (A375, P<0.0032; MUM-2B, P<0.0005;MUM-2C, P<0.0059) (FIG. 3e,f ). In aggregate, these findings show inrelevant xenograft models of early melanoma development theparticipation of ABCB5⁺ MMIC in the genesis of vasculogenic channels,and the interdependency of MMIC-derived channel formation and tumourgrowth.

Discovery of MMIC-driven vasculogenesis identifies selectivedifferentiation plasticity as a novel CSC-specific function throughwhich these tumourigenic cancer subpopulations may provide a specificgrowth advantage to developing tumours. Our finding of a propensity ofMMIC to differentiate selectively into cells capable of serving adefined tissue function required for more efficient tumour growthparallels hallmark characteristics of physiological stem cells, whichsimilarly give rise to cell lineages capable of serving specific rolesrequired for maintenance of tissue homeostasis through defineddifferentiation programs. Importantly, we find that MMIC-dependenttumourigenesis and vasculogenesis are operative not only in humanmelanoma to murine skin xenotransplantation models but also upon humanmelanoma to human skin transplantation. Therefore, our results provideinitial evidence that the tumour-sustaining role of human CSC identifiedin xenotransplantation assays does not merely reflect the limitedability of human tumour cells to adapt to growth in a foreign (mouse)milieu, as has been postulated based on the results of murine tumourtransplantation experiments utilizing histocompatible murine hosts¹⁷

The now widely-accepted concept of cancer angiogenesis advanced byFolkman in 1971 states that human cancers are critically dependent upontumour-related blood-vessel growth and development¹⁸. In addition toclassical angiogenesis whereby cancer cells, including CSC¹⁹, inducein-growth of mature, CD31-positive vessels from surrounding stroma²⁰,evidence has been generated that cancer cells may also directly formsurrogate vessel-like spaces by the process of vasculogenic mimicrywhereby aggressive human melanomas develop patterned networks composedof periodic acid-Schiff (PAS)- and laminin-reactive basement membranesand associated perfusable channels formed by tumour cells that expresssome but not all endothelium-related genes and proteins⁴. The presentstudy identifies the cells and underlying mechanisms responsible forvasculogenic mimicry, and establishes that in addition to self-renewal,MMIC selectively express vasculogenic genes and form channels consistentwith the function of promoting nutrition to rapidly growing tumours.Thus, cancer angiogenesis and MMIC-driven vasculogenesis may representindependent yet potentially interrelated mechanisms whereby aggressiveand metabolically-active tumours obtain those nutrients requisite forcritical stages of growth and evolution. This may be particularlyimportant during tumour initiation and early phases of tumourigenicgrowth when hypoxia-dependent, mTOR-driven angiogenesis from surroundingstroma has not fully evolved²¹.

Recently, proof-of-principle has been established for the potentialtherapeutic utility of the CSC concept^(3,22). Therefore, identificationof a vasculogenic mechanism whereby MMIC may contribute to tumour growthhas potentially important therapeutic implications. Previous studiesrevealed that normally resistant human melanoma cells are renderedsensitive in vitro to the effects of chemotherapeutic agents by mAb- orsiRNA-mediated blockade of ABCB5^(5,6), and that mAb binding to ABCB5 issufficient to induce an effective anti-melanoma immune response viaantibody-dependent cell-mediated cytotoxicity (ADCC) in vivo³. Now,recognition of the role of MMIC in tumour vasculogenesis will alsopermit development of strategies focused on inhibition of the relevantmolecular pathways integral to endothelial-directed CSC plasticity.Moreover, the spatial localization of the MMIC component to channelsthat communicate with the systemic circulation may render this importantdeterminant of cancer virulence particularly vulnerable to therapeutictargeting.

Example 5: Vasculogenic/Angiogenic Pathways in Human Melanoma

We investigated gene relationships based on Ingenuity Pathway Analysis.We prepared a graphical representation of pathway activation acrossABCB5⁺ MMIC. Genes that were overexpressed in ABCB5⁺ relative to ABCB5⁻human melanoma cells were represented by red nodes (circles) and thoseexpressed at lower levels were represented by black nodes. Black lineswere drawn between genes to show known interactions. Known genefunctions in vasculogenesis and angiogenesis, and genes known asrelevant drug targets were annotated (red lines) (FIG. 1a ). We examinedexpression of vasculogenic/angiogenic pathway members by RT-PCR inABCB5⁺ MMIC. Results of this anaylsis are shown in FIG. 1b . We useddual color flow cytometry using ABCB5 phenotype-specific cell gating todetermining FLT1 (VEGFR-1) protein expression of ABCB5⁺ MMIC (FIG. 1c ,top) and ABCB5-melanoma cells (FIG. 1c , bottom). We examined CD144expression in ABCB5⁺ MMIC or ABCB5⁻ melanoma cell subpopulations byimmunofluorescence staining prior to (t=0 h) and upon 48 h of culture(t=48 h) in the presence of 100 ng/ml VEGF¹¹. Representativeimmunofluorescence staining for CD144 expression (Texas red) are shownin FIG. 1d , with nuclei counterstained in blue (DAPI). Mean percentages(mean±s.e.m., n=3 replicate experiments) of cells staining positivelyfor CD144 in each sample are shown on the right. We examined CD144expression in melanoma cells in the presence of 100 ng/ml VEGF as inabove, but in the presence or absence of anti-FLT1 (VEGFR-1) blockingmAb or isotype control mAb. Representative immunofluorescence stainingfor CD144 expression (Texas red) by melanoma cells cultured for 48 h(t=48 h) are shown in FIG. 1e , with nuclei counterstained in blue(DAPI). Mean percentages (mean±s.e.m., n=3 replicate experiments) ofcells staining positively for CD144 in each sample are shown in the farright panel. We examined tube formation by phase contrast lightmicroscopy of melanoma cells cultured for 24 h (t=24 h) in the presenceof 100 ng/ml VEGF and the presence or absence of anti-FLT1 (VEGFR-1)blocking mAb or isotype control mAb (FIG. 1f ). Number oftubes/microscopy field (mean±s.e.m., n=3 replicate experiments) and tubelength (μm) (mean±s.e.m., n=3 replicate experiments) are illustrated forthe different experimental conditions on the far right panels,respectively. We examined the differentiation potential of ABCB5⁺ andABCB5⁻ human melanoma cells along a adipogenic pathway (FIG. 1h , OilRed O staining, nuclei are counterstained with hematoxylin) andosteogenic pathway (FIG. 1i , Alizarin Red staining). Myogenicdifferentiation potential of ABCB5⁺ and ABCB5⁻ human melanoma cells wasalso examined (FIG. 1j ). Absence of myogenin staining (FITC, green) wasdetected in ABCB5⁺ or ABCB5⁻ human melanoma cells (nuclei arecounterstained with DAPI).

Example 6: MMIC-Driven In Vivo Vasculogenesis

We investigated MMIC driven vasculogenesis in vivo. Sections of humanmelanoma growing at melanoma cell injection site within human dermis ofskin xenograft to NOD/SCID mouse were conventionally-stained byhematoxylin and eosin (FIG. 2a ). We also examined byimmunohistochemistry the expression of human CD31 which indicatedangiogenic response at perimeter of melanoma within human xenograft.(FIG. 2b , broken line represents interface of tumour nodule with dermalconnective tissue). We used periodic-acid Schiff (PAS) stain (withdiastase), an immunochemical stain of CD31-negative interior regions ofmelanoma xenografts, to reveal numerous anastomosing channels (FIG. 2c ,inset is laminin immunohistochemistry indicating identical pattern). Weconducted transmission electron micrographs of interior regions ofmelanoma xenografts (FIG. 2d ), and found that lumenal spaces containingblood products (erythrocytes) are lined by melanoma cells and associatedbasement membrane-like extracellular matrix. We examined the interiorzone of melanoma xenograft derived from cells expressing GFP transgeneand immunohistochemically stained for endothelial marker CD144 (redchromogen); results are shown in (FIG. 2e ). We found that CD144expression is confined to cells forming lumen-like spaces lined by cellsthat co-express GFP and CD144 (indicated as yellow-orange). We alsoperformed immunohistochemistry, at low (FIG. 2f ) and high (FIG. 2g )magnification, for ABCB5 protein; our results show that reactivity isrestricted to anastomosing channels identical to those seen in FIG. 2c .The inset in FIG. 2f depicts similar formation of ABCB5-reactivechannels in a patient-derived melanoma biopsy. We performed in situhybridization for ABCB5 mRNA (FIG. 2h ). Our results reveal a channelpattern corresponding to that of ABCB5 protein expression (compare withFIG. 2f ; inset is sense control). We examined the expression of ABCB5in melanoma xenografts after intravenous administration in vivo (FIG. 2h). Detection of anti-ABCB5 mAb was accomplished using anti-mouse Igimmunohistochemistry; note localization to channels (inset representsanti-mouse Ig staining after intravenous administration of irrelevantisotype-matched control mAb). Dual-labeling immunofluorescencemicroscopy was performed for both ABCB5 (green), CD144 (red), and ABCB5& CD144 (mix) (FIG. 2j ) and ABCB5 (green), TIE-1 (red), and ABCB5 &TIE-1 (mix) (FIG. 2j ).

Example 7: Interdependency of MMIC-Driven Vasculogenesis andTumourigenesis

We examined ABCB5 expression by flow cytometry; ABCB5 or controlstaining (FITC, F11) was plotted against forward scatter (FSC) for humanA375, MUM-2B, and MUM-2C melanoma cell inocula. Representative data isshown in FIG. 3a . We examined histologic sections of melanomas thatdeveloped from three unsegregated and ABCB5-depleted melanoma cell linesinjected intradermally into human skin xenografts. Representativesections are shown in FIG. 3b . We used histology to determine tumourformation rate (%) 3 weeks following intradermal transplantation ofunsegregated vs. ABCB5⁺-depleted human A375, MUM-2B or MUM-2C melanomacells (2×10⁶/inoculum) into human skin/Rag2^(−/−) chimeric mice (n=5,respectively). (FIG. 3c ). We determined histological tumour volumes(mean±s.e.m.) 3 weeks following intradermal transplantation ofunsegregated vs. ABCB5⁺-depleted human A375, MUM-2B or MUM-2C melanomacells (2×10⁶/inoculum) into human skin/Rag2^(−/−) chimeric mice. (FIG.3d ). We performed immunohistochemistry for laminin. Our results showedthe extent of channel formation in melanomas that developed fromunsegregated or ABCB5⁺-depleted melanoma cell inocula derived from A375,MUM-2B or MUM-2C lines injected intradermally into human skin xenografts(arrows=necrosis). (FIG. 3e ). We performed image analysis of lamininimmunoreactivity for melanomas derived from unsegregated andABCB5⁺-depleted cell inocula. Data are shown in FIG. 3f ; y-axis ispercent of pixelated area with reactivity (mean±s.e.m.); solid barrepresents tumours derived from unsegregated melanoma cells, open barsrepresent tumours derived from ABCB5⁺-depleted cells (A375, P<0.0032;MUM-2B, P<0.0005; MUM-2C, P<0.0059).

Example 8: Correlation of ABCB5 Protein and mRNA Expression Across HumanMelanoma Cell Lines

We examined ABCB5 and tubulin expression in a panel of human melanomacell lines by western blot analysis (FIG. 4a ). We examined relativeABCB5 mRNA expression (log 2) in a panel of human melanoma cell linesplotted against ABCB5 protein expression as determined by ratios ofABCB5 89 kD western blot band intensity and tubulin western blot bandintensity for each human melanoma cell line. (FIG. 4b ). Data points areas follows: 1, SK-MEL-2; 2, SK-MEL-5; 3, SK-MEL-28; 4, MDA-MB-435; 5,UACC-62; 6, UACC-257; 7, M14; 8, MALME-3M. Spearman Rank Correlation r(corrected for ties).

Example 9: CSC-Associated Genes Identified at the Protein Level

Using cell surface immunostaining and flow cytometry we identifiedadditional genes to be differentially regulated at the protein level inABCB5+ CSC versus ABCB5− cancer bulk populations. These are allimmunomodulatory molecules and the ones upregulated in ABCB5+ cells maybe relevant to the escape from immunosurveillance and be resposible forresistance to immunotherapy in malignant melanoma, i.e. when the genesoverexpressed on ABCB5+ cells are targeted, melanoma is predicted to besensitized to immune attack and therapy.

TABLE 8 Upregulated in ABCB5+ CSC compared to ABCB5− bulk cancerpopulations: MHC class II CD28 CD86 PD-1 CD40-L 4-1BB-L B7-H4 GITR

TABLE 7 Downregulated in ABCB5+ CSC compared to ABCB5− cancer bulkpopulations MHC class I CD80 PD-L1 ICOS-L

REFERENCES FOR DETAILED DESCRIPTION AND EXAMPLES

-   1 M. Al-Hajj, M. S. Wicha, A. Benito-Hernandez et al., Proc Natl    Acad Sci USA 100 (7), 3983 (2003); D. Bonnet and J. E. Dick, Nat Med    3 (7), 730 (1997); C. A. O'Brien, A. Pollett, S. Gallinger et al.,    Nature 445 (7123), 106 (2007); L. Ricci-Vitiani, D. G. Lombardi, E.    Pilozzi et al., Nature 445 (7123), 111 (2007); S. K. Singh, C.    Hawkins, I. D. Clarke et al., Nature 432 (7015), 396 (2004).-   2 T. Schatton and M. H. Frank, Pigment cell & melanoma research 21    (1), 39 (2008).-   3 T. Schatton, G. F. Murphy, N. Y. Frank et al., Nature 451 (7176),    345 (2008).-   4 A. J. Maniotis, R. Folberg, A. Hess et al., Am J Pathol 155 (3),    739 (1999).-   5 N. Y. Frank, A. Margaryan, Y. Huang et al., Cancer Res 65 (10),    4320 (2005).-   6 Y. Huang, P. Anderle, K. J. Bussey et al., Cancer Res 64 (12),    4294 (2004).-   7 L. Chin, L. A. Garraway, and D. E. Fisher, Genes Dev 20 (16), 2149    (2006).-   8 G. I. Keshet, I. Goldstein, O. Itzhaki et al., Biochem Biophys Res    Commun 368 (4), 930 (2008).-   9 J. F. Sousa and E. M. Espreafico, BMC cancer 8, 19 (2008).-   10 N. Y. Frank, A. T. Kho, T. Schatton et al., J Cell Biol 175 (1),    99 (2006).-   11 K. Nishiyama, K. Takaji, Y. Uchijima et al., J Biol Chem 282    (23), 17200 (2007).-   12 D. Fang, T. K. Nguyen, K. Leishear et al., Cancer Res 65 (20),    9328 (2005).-   13 I. Juhasz, S. M. Albelda, D. E. Elder et al., Am J Pathol 143    (2), 528 (1993).-   14 E. Monzani, F. Facchetti, E. Galmozzi et al., Eur J Cancer 43    (5), 935 (2007).-   15 W. M. Klein, B. P. Wu, S. Zhao et al., Mod Pathol 20 (1), 102    (2007).-   16 E. A. Seftor, P. S. Meltzer, D. A. Kirschmann et al., Clin Exp    Metastasis 19 (3), 233 (2002).-   17 P. N. Kelly, A. Dakic, J. M. Adams et al., Science 317 (5836),    337 (2007).-   18 J. Folkman, N Engl J Med 285 (21), 1182 (1971).-   19 S. Bao, Q. Wu, S. Sathornsumetee et al., Cancer Res 66 (16), 7843    (2006); R. J. Gilbertson and J. N. Rich, Nat Rev Cancer 7 (10), 733    (2007).-   20 B. N. Perry and J. L. Arbiser, J Invest Dermatol 126 (10), 2160    (2006).-   21 M. Guba, P. von Breitenbuch, M. Steinbauer et al., Nat Med 8 (2),    128 (2002).-   22 Z. F. Yang, D. W. Ho, M. N. Ng et al., Cancer Cell 13 (2), 153    (2008); S. Bao, Q. Wu, Z. Li et al., Cancer Res 68 (15), 6043    (2008).-   23 N. Y. Frank, S. S. Pendse, P. H. Lapchak et al., J Biol Chem 278    (47), 47156 (2003).-   24 D. Donovan, N. J. Brown, E. T. Bishop et al., Angiogenesis 4 (2),    113 (2001).

Having thus described several aspects of at least one embodiment of thisinvention, it is to be appreciated various alterations, modifications,and improvements will readily occur to those skilled in the art. Suchalterations, modifications, and improvements are intended to be part ofthis disclosure, and are intended to be within the spirit and scope ofthe invention. Accordingly, the foregoing description and drawings areby way of example only.

1. A method for diagnosing cancer in an individual, comprising:determining an expression level of a cancer stem cell (CSC)-associatedgene in Table 5 in a test sample from the individual; and comparing theexpression level of the CSC-associated gene to a reference value,wherein results of the comparison are diagnostic of cancer. 2-21.(canceled)
 22. A method for isolating a cancer stem cell, comprising:contacting a sample with an agent that binds a polypeptide encoded by aCSC-associated gene in Table 4, wherein the polypeptide is expressed onthe surface of the cancer stem cell, and wherein, if the sample containsthe cancer stem cell, the agent binds to the polypeptide; and isolatingthe agent from the sample. 23-31. (canceled)
 32. A method for treatingan individual having, or at risk of having, cancer, comprising:administering a therapeutically effective amount of a composition thatinduces the expression of a CSC-associated gene selected from the groupset forth in Table 2 or
 7. 33-42. (canceled)
 43. A method for treatingan individual having, or at risk of having, cancer, comprising:administering a therapeutically effective amount of a composition thattargets a product of a CSC-associated gene selected from the group setforth in Table 1 or
 8. 44-65. (canceled)
 66. A method of delivering atherapeutic agent to a cancer stem cell, comprising: contacting a cancerstem cell with an isolated molecule that selectively binds to apolypeptide encoded by a CSC-associated gene selected from the group setforth in Table 4 conjugated to a therapeutic agent in an effectiveamount to deliver the therapeutic agent to the cancer stem cell. 67-75.(canceled)
 76. An isolated molecule that selectively binds to apolypeptide encoded by a CSC-associated gene set forth in Table 4, andthat is conjugated to a therapeutic agent.
 77. The isolated molecule ofclaim 76, wherein the CSC-associated gene is selected from the groupconsisting of: ANK2, NCKAP1L, PTPRE, PTPRS, SBF1, SCN3A, SGCA, SGCB,SLC2A11, SLC2A8, SLC4A1, STX3, and TBC1D8.
 78. The isolated molecule ofclaim 76, wherein the therapeutic agent is selected from: a toxin, asmall-interfering nucleic acid, and a chemotherapeutic agent.
 79. Theisolated molecule of claim 76, wherein the isolated molecule is anantibody or antigen-binding fragment.
 80. The isolated molecule of claim76, wherein the isolated molecule is an isolated receptor ligand of thepolypeptide.
 81. The isolated molecule of claim 79, wherein the antibodyor antigen-binding fragment is a monoclonal antibody, polyclonalantibody, human antibody, chimeric antibody, humanized antibody,single-chain antibody, a F(ab′)₂, Fab, Fd, or Fv fragment. 82-84.(canceled)